WO2006078992A2 - Heteroaryl substituted piperazinyl-pyridine analogues - Google Patents

Heteroaryl substituted piperazinyl-pyridine analogues Download PDF

Info

Publication number
WO2006078992A2
WO2006078992A2 PCT/US2006/002194 US2006002194W WO2006078992A2 WO 2006078992 A2 WO2006078992 A2 WO 2006078992A2 US 2006002194 W US2006002194 W US 2006002194W WO 2006078992 A2 WO2006078992 A2 WO 2006078992A2
Authority
WO
WIPO (PCT)
Prior art keywords
alkyl
compound
pain
patient
halogen
Prior art date
Application number
PCT/US2006/002194
Other languages
French (fr)
Other versions
WO2006078992A3 (en
Inventor
Charles A. Blum
Original Assignee
Neurogen Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Neurogen Corporation filed Critical Neurogen Corporation
Priority to CA002595205A priority Critical patent/CA2595205A1/en
Priority to EP06733793A priority patent/EP1838703A2/en
Priority to JP2007552316A priority patent/JP2008528506A/en
Priority to AU2006206246A priority patent/AU2006206246A1/en
Priority to US11/795,651 priority patent/US20080124384A1/en
Publication of WO2006078992A2 publication Critical patent/WO2006078992A2/en
Publication of WO2006078992A3 publication Critical patent/WO2006078992A3/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/10—Drugs for disorders of the urinary system of the bladder
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/04—Antipruritics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/04—Centrally acting analgesics, e.g. opioids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings

Definitions

  • This invention relates generally to heteroaryl substituted piperazinyl-pyridine analogues that have useful pharmacological properties.
  • the invention further relates to the use of such compounds for treating conditions related to capsaicin receptor activation, for identifying other agents that bind to capsaicin receptor, and as probes for the detection and localization of capsaicin receptors.
  • Pain perception, or nociception is mediated by the peripheral terminals of a group of specialized sensory neurons, termed "nociceptors.”
  • nociceptors A wide variety of physical and chemical stimuli induce activation of such neurons in mammals, leading to recognition of a potentially harmful stimulus. Inappropriate or excessive activation of nociceptors, however, can result in debilitating acute or chronic pain.
  • Neuropathic pain involves pain signal transmission in the absence of stimulus, and typically results from damage to the nervous system. In most instances, such pain is thought to occur because of sensitization in the peripheral and central nervous systems following initial damage to the peripheral system (e.g., via direct injury or systemic disease). Neuropathic pain is typically burning, shooting and unrelenting in its intensity and can sometimes be more debilitating that the initial injury or disease process that induced it.
  • neuropathic pain is frequently non-responsive or only partially responsive to conventional opioid analgesic regimens.
  • Treatments employing the N-methyl-D-aspartate antagonist ketamine or the alpha(2)-adrenergic agonist clonidine can reduce acute or chronic pain, and permit a reduction in opioid consumption, but these agents are often poorly tolerated due to side effects.
  • Topical treatment with capsaicin has been used to treat chronic and acute pain, including neuropathic pain.
  • Capsaicin is a pungent substance derived from the plants of the Solanaceae family (which includes hot chili peppers) and appears to act selectively on the small diameter afferent nerve fibers (A-delta and C fibers) that are believed to mediate pain.
  • the response to capsaicin is characterized by persistent activation of nociceptors in peripheral tissues, followed by eventual desensitization of peripheral nociceptors to one or more stimuli. From studies in animals, capsaicin appears to trigger C fiber membrane depolarization by opening cation selective channels for calcium and sodium.
  • Similar responses are also evoked by structural analogues of capsaicin that share a common vanilloid moiety.
  • One such analogue is resiniferatoxin (RTX), a natural product of Euphorbia plants.
  • RTX resiniferatoxin
  • VR vanilloid receptor
  • the capsaicin response is competitively inhibited (and thereby antagonized) by another capsaicin analog, capsazepine, and is also inhibited by the non-selective cation channel blocker ruthenium red, which binds to VR with no more than moderate affinity (typically with a K; value of no lower than 140 ⁇ M).
  • Rat and human vanilloid receptors have been cloned from dorsal root ganglion cells.
  • the first type of vanilloid receptor to be identified is known as vanilloid receptor type 1 (VRl), and the terms "VRl” and “capsaicin receptor” are used interchangeably herein to refer to rat and/or human receptors of this type, as well as mammalian homologues.
  • VRl vanilloid receptor type 1
  • the role of VRl in pain sensation has been confirmed using mice lacking this receptor, which exhibit no vanilloid-evoked pain behavior and impaired responses to heat and inflammation.
  • VRl is a nonselective cation channel with a threshold for opening that is lowered in response to elevated temperatures, low pH, and capsaicin receptor agonists.
  • Opening of the capsaicin receptor channel is generally followed by the release of inflammatory peptides from neurons expressing the receptor and other nearby neurons, increasing the pain response.
  • the capsaicin receptor undergoes a rapid desensitization via phosphorylation by cAMP-dependent protein kinase.
  • VRl agonist vanilloid compounds Because of their ability to desensitize nociceptors in peripheral tissues, VRl agonist vanilloid compounds have been used as topical anesthetics. However, agonist application may itself cause burning pain, which limits this therapeutic use. Recently, it has been reported that VRl antagonists, including certain nonvanilloid compounds, are also useful for the treatment of pain (see, e.g., PCT International Application Publication Numbers WO 02/08221, WO 03/062209, WO 04/054582, WO 04/055003, WO 04/055004, WO 04/056774, WO 05/007646, WO 05/007648, WO 05/007652, WO 05/009977, WO 05/009980 and WO 05/009982).
  • the present invention provides heteroaryl substituted piperazinyl-pyridine analogues of Formula I:
  • a ⁇ i is a 5-membered aromatic heterocycle that is substituted with from O to 4 substituents independently chosen from Ri;
  • Ar 2 is phenyl or a 6-membered aromatic heterocycle, each of which is optionally substituted, and is preferably substituted with from O to 4 substituents independently chosen from R 2 ;
  • W is CH or N;
  • X, Y and Z are independently CR x or N, such that at least one of X, Y and Z is N;
  • R x is independently chosen at each occurrence from hydrogen, Ci-Qalkyl, amino, cyano and mono- and di-(Ci-C 4 alkyl)amino;
  • Each Ri is independently chosen from:
  • N(R 2 )S(O) n S(O) m N(R z ) and N[S(O) m R 2 ]S(O) m ; wherein m is independently selected at each occurrence from O, 1 and 2; and R 2 is independently selected at each occurrence from hydrogen, C]-C 8 alkyl and groups that are taken together with R y to form an optionally substituted 4- to 7-membered heterocycle; and
  • Each Ry is independently hydrogen, C]-C 8 haloalkyl, optionally substituted Ci-C 8 alkyl, optionally substituted (C 3 -C 8 carbocycle)C 0 -C 4 alkyl, optionally substituted (4- to 7-membered heterocycle)Co-C 4 alkyl, or taken together with R 2 to form an optionally substituted 4- to 7- membered heterocycle, wherein each alkyl, carbocycle and heterocycle is preferably substituted with from 0 to 4 substituents independently selected from hydroxy, halogen, amino, cyano, nitro, oxo, -COOH, aminocarbonyl, C r C 6 alkyl, C 3 -C 7 cycloalkyl, C 2 -C 6 alkyl ether, C r C 6 alkanoyl, -SO 2 (C r C 6 alkyl), -SO 2 NH 2 , C r C 8 alkoxy, Ci-C 8 alkylthio, mono- and di
  • C 6 alkyl)aminocarbonyl each of which is optionally substituted, and is preferably substituted with from O to 4 substituents independently chosen from halogen, hydroxy, cyano, amino, -COOH and oxo; or
  • R 3 is selected from:
  • L is Co-C ⁇ alkylene or Ci-C 6 alkyl that is taken together with R 5 , R 6 or R 7 to form a 4- to 7- membered heterocycle;
  • W is O, CO, S, SO or SO 2 ;
  • R 5 and R 6 are:
  • R 7 is hydrogen, C r Ci 2 alkyl, C 2 -C ]2 alkenyl, (C 3 -C 8 cycloalkyl)C 0 -C 4 alkyl, C 2 -C 6 alkanoyl, phenylCo-C 6 alkyl, (4- to 7-membered heterocycle)Co-C 6 alkyl or a group that is joined to L to form a 4- to 7-membered heterocycle; wherein each of (ii) and (iii) is optionally substituted, and is preferably substituted with from 0 to 4 substituents independently chosen from:
  • R 4 represents from 0 to 2 substituents that are preferably independently chosen from Ci-C 3 alkyl,
  • compounds of Formula I are VRl modulators and exhibit a Kj of no greater than 1 micromolar, 500 nanomolar, 100 nanomolar, 50 nanomolar, 10 nanomolar or 1 nanomolar in a capsaicin receptor binding assay and/or have an EC 50 or IC 50 value of no greater than 1 micromolar, 500 nanomolar, 100 nanomolar, 50 nanomolar, 10 nanomolar or 1 nanomolar in an in vitro assay for determination of capsaicin receptor agonist or antagonist activity.
  • VRl modulators are VRl antagonists and exhibit no detectable agonist activity in an in vitro assay of capsaicin receptor activation (e.g., the assay provided in Example 6, herein) at a concentration equal to the IC 50 , 10 times the IC 50 or 100 times the IC 50 .
  • compounds provided herein are labeled with a detectable marker (e.g., radiolabeled or fluorescein conjugated).
  • a detectable marker e.g., radiolabeled or fluorescein conjugated.
  • the present invention further provides, within other aspects, pharmaceutical compositions comprising at least one compound of Formula I in combination with a physiologically acceptable carrier or excipient.
  • methods for reducing calcium conductance of a cellular capsaicin receptor, comprising contacting a cell (e.g., neuronal, such as cells of the central nervous system and/or peripheral ganglia, urothelial or lung) that expresses a capsaicin receptor with at least one VRl modulator as described herein.
  • a cell e.g., neuronal, such as cells of the central nervous system and/or peripheral ganglia, urothelial or lung
  • Such contact may occur in vivo or in vitro and is generally performed using a concentration of VRl modulator that is sufficient to alter the binding of vanilloid ligand to VRl in vitro (using the assay provided in Example 5) and/or VRl- mediated signal transduction (using an assay provided in Example 6).
  • Methods are further provided for inhibiting binding of vanilloid ligand to a capsaicin receptor.
  • the inhibition takes place in vitro.
  • Such methods comprise contacting a capsaicin receptor with at least one VRl modulator as described herein, under conditions and in an amount or concentration sufficient to detectably inhibit vanilloid ligand binding to the capsaicin receptor.
  • the capsaicin receptor is in a patient.
  • Such methods comprise contacting cells expressing a capsaicin receptor in a patient with at least one VRl modulator as described herein in an amount or concentration that would be sufficient to detectably inhibit vanilloid ligand binding to cells expressing a cloned capsaicin receptor in vitro.
  • the present invention further provides methods for treating a condition responsive to capsaicin receptor modulation in a patient, comprising administering to the patient a therapeutically effective amount of at least one VRl modulator as described herein.
  • methods for treating pain in a patient, comprising administering to a patient suffering from (or at risk for) pain a therapeutically effective amount of at least one VRl modulator as described herein.
  • Methods are further provided for treating itch, urinary incontinence, overactive bladder, cough and/or hiccup in a patient, comprising administering to a patient suffering from (or at risk for) one or more of the foregoing conditions a therapeutically effective amount of at least one VRl modulator as described herein.
  • the present invention further provides methods for promoting weight loss in an obese patient, comprising administering to an obese patient a therapeutically effective amount of at least one VRl modulator as described herein.
  • Methods are further provided for identifying an agent that binds to capsaicin receptor, comprising: (a) contacting capsaicin receptor with a labeled compound as described herein under conditions that permit binding of the compound to capsaicin receptor, thereby generating bound, labeled compound; (b) detecting a signal that corresponds to the amount of bound, labeled compound in the absence of test agent; (c) contacting the bound, labeled compound with a test agent; (d) detecting a signal that corresponds to the amount of bound labeled compound in the presence of test agent; and (e) detecting a decrease in signal detected in step (d), as compared to the signal detected in step (b).
  • the present invention provides methods for determining the presence or absence of capsaicin receptor in a sample, comprising: (a) contacting a sample with a compound as described herein under conditions that permit binding of the compound to capsaicin receptor; and (b) detecting a signal indicative of a level of the compound bound to capsaicin receptor.
  • the present invention also provides packaged pharmaceutical preparations, comprising: (a) a pharmaceutical composition as described herein in a container; and (b) instructions for using the composition to treat one or more conditions responsive to capsaicin receptor modulation, such as pain, itch, urinary incontinence, overactive bladder, cough, hiccup and/or obesity.
  • the present invention provides methods for preparing the compounds disclosed herein, including the intermediates.
  • the present invention provides heteroaryl substituted piperazinyl-pyridine analogues. Such compounds may be used in vitro or in vivo, to modulate capsaicin receptor activity in a variety of contexts.
  • heteroaryl substituted piperazinyl-pyridine analogue encompasses all compounds of Formula I, as well as compounds of other Formulas provided herein (including any enantiomers, racemates and stereoisomers) and pharmaceutically acceptable salts of such compounds.
  • compounds in which the core ring encompasses all compounds of Formula I, as well as compounds of other Formulas provided herein (including any enantiomers, racemates and stereoisomers) and pharmaceutically acceptable salts of such compounds.
  • compounds in which the core ring encompasses all compounds of Formula I, as well as compounds of other Formulas provided herein (including any enantiomers, racemates and stereoisomers) and pharmaceutically acceptable salts of such compounds.
  • compounds in which the core ring encompasses all compounds of Formula I, as well as compounds of other Formulas provided herein (including any enantiomers, racemates and stereoisomers) and pharmaceutically acceptable salts of such compounds.
  • compounds in which the core ring encompasses all compounds of Formula I, as well as compounds of
  • x R 3 is pyridyl, pyrimidyl or triazinyl ⁇ e.g.,
  • a "pharmaceutically acceptable salt” of a compound is an acid or base salt that is generally considered in the art to be suitable for use in contact with the tissues of human beings or animals without excessive toxicity or carcinogenicity, and preferably without irritation, allergic response, or other problem or complication.
  • Such salts include mineral and organic acid salts of basic residues such as amines, as well as alkali or organic salts of acidic residues such as carboxylic acids.
  • Specific pharmaceutical salts include, but are not limited to, salts of acids such as hydrochloric, phosphoric, hydrobromic, malic, glycolic, fumaric, sulfuric, sulfamic, sulfanilic, formic, toluenesulfonic, methanesulfonic, benzene sulfonic, ethane disulfonic, 2- hydroxyethylsulfonic, nitric, benzoic, 2-acetoxybenzoic, citric, tartaric, lactic, stearic, salicylic, glutamic, ascorbic, pamoic, succinic, fumaric, maleic, propionic, hydroxymaleic, hydroiodic, phenylacetic, alkanoic such as acetic, HOOC-(CH 2 ) n -COOH where n is 0-4, and the like.
  • acids such as hydrochloric, phosphoric, hydrobromic, malic, glycolic, fumaric, sulfur
  • pharmaceutically acceptable cations include, but are not limited to sodium, potassium, calcium, aluminum, lithium and ammonium.
  • pharmaceutically acceptable salts for the compounds provided herein, including those listed within Remington: The Science and Practice of Pharmacy, 21 st ed., Lippincott Williams & Wilkins, Philadelphia, PA (2005).
  • a pharmaceutically acceptable acid or base salt can be synthesized from a parent compound that contains a basic or acidic moiety by any conventional chemical method.
  • such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, the use of nonaqueous media, such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile, is preferred.
  • nonaqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile
  • prodrugs of the compounds of Formula I are provided herein.
  • a "prodrug” is a compound that may not fully satisfy the structural requirements of the compounds provided herein, but is modified in vivo, following administration to a patient, to produce a compound of Formula I, or other formula provided herein.
  • a prodrug may be an acylated derivative of a compound as provided herein.
  • Prodrugs include compounds wherein hydroxy, amine or sulfhydryl groups are bonded to any group that, when administered to a mammalian subject, cleaves to form a free hydroxy, amino or sulfhydryl group, respectively.
  • Examples of prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of alcohol and amine functional groups within the compounds provided herein.
  • Prodrugs of the compounds provided herein may be prepared by modifying functional groups present in the compounds in such a way that the modifications are cleaved in vivo to yield the parent compounds.
  • alkyl refers to a straight or branched chain saturated aliphatic hydrocarbon.
  • Alkyl groups include groups having from 1 to 8 carbon atoms (C r C 8 alkyl), from 1 to 6 carbon atoms (Q-C ⁇ alkyl) and from 1 to 4 carbon atoms (Ci-C 4 alkyl), such as methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, 2-pentyl, isopentyl, neopentyl, hexyl, 2- hexyl, 3-hexyl and 3-methylpentyl.
  • Co-C n alkyl refers to a single covalent bond (C 0 ) or an alkyl group having from 1 to n carbon atoms; for example "Co-C 4 alkyl” refers to a single covalent bond or a Ci-C 4 alkyl group; “C 0 -C 8 alkyl” refers to a single covalent bond or a d-C 8 alkyl group. In some instances, a substituent of an alkyl group is specifically indicated.
  • hydroxyalkyl refers to an alkyl group substituted with at least one hydroxy substituent ⁇ e.g., "C 1 - C ⁇ ydroxyalkyl” refers to a Ci-C 4 alkyl group that has at least one -OH substituent).
  • Alkylene refers to a divalent alkyl group, as defined above.
  • Co-C 4 alkylene is a single covalent bond or an alkylene group having 1, 2, 3 or 4 carbon atoms;
  • Cj-Qalkylene is an alkylene group having from 1 to 4 carbon atoms;
  • C r C 6 alkylene is an alkylene group having from 1 to 6 carbon atoms.
  • Alkenyl refers to straight or branched chain alkene groups, which comprise at least one unsaturated carbon-carbon double bond. Alkenyl groups include C 2 -C 8 alkenyl, C 2 -C 6 alkenyl and C 2 -C 4 alkenyl groups, which have from 2 to 8, 2 to 6 or 2 to 4 carbon atoms, respectively, such as ethenyl, allyl or isopropenyl. "Alkynyl” refers to straight or branched chain alkyne groups, which have one or more unsaturated carbon-carbon bonds, at least one of which is a triple bond.
  • Alkynyl groups include C 2 -C 8 alkynyl, C 2 -C 6 alkynyl and C 2 -C 4 alkynyl groups, which have from 2 to 8, 2 to 6 or 2 to 4 carbon atoms, respectively.
  • a “cycloalkyl” is a group that comprises one or more saturated and/or partially saturated rings in which all ring members are carbon, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, adamantyl, decahydro-naphthalenyl, octahydro-indenyl, and partially saturated variants of the foregoing, such as cyclohexenyl. Cycloalkyl groups do not comprise an aromatic ring or a heterocyclic ring.
  • cycloalkyl groups are C 3 -C 8 cycloalkyl, in which the group contains a single ring having from 3 to 8 ring members, all of which are carbon.
  • a "(C 3 -C 8 cycloalkyl)Co-C 4 alkyr T is a 3- to 8-membered cycloalkyl group linked via a single covalent bond or a Cj-Qalkylene group.
  • alkoxy is meant an alkyl group as described above attached via an oxygen bridge.
  • Alkoxy groups include Ci-C 6 alkoxy and C r C 4 alkoxy groups, which have from 1 to 6 or from 1 to 4 carbon atoms, respectively.
  • Methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, see-butoxy, tert-buXoxy, n-pentoxy, 2-pentoxy, 3-pentoxy, isopentoxy, neopentoxy, hexoxy, 2- hexoxy, 3-hexoxy, and 3-methyl ⁇ entoxy are representative alkoxy groups.
  • alkylthio refers to an alkyl group as described above attached via a sulfur bridge.
  • aminoalkyl refers to an alkyl group as described above substituted with an imine ⁇ e.g., a group of the formula
  • Alkanoyl groups have the indicated number of carbon atoms, with the carbon of the keto group being included in the numbered carbon atoms.
  • Alkanoyl groups include, for example, C 2 - Qalkanoyl, C 2 -C 6 alkanoyl and C 2 -C 4 alkanoyl groups, which have from 2 to 8, from 2 to 6 or from 2 to 4 carbon atoms, respectively.
  • alkanone is a ketone group in which carbon atoms are in a linear or branched alkyl arrangement.
  • C 3 -C 8 alkanone refers to an alkanone having from 3 to 8, 6 or 4 carbon atoms, respectively.
  • alkyl ether refers to a linear or branched ether substituent (i.e., an alkyl group that is substituted with an alkoxy group).
  • Alkyl ether groups include C 2 -C 8 alkyl ether, C 2 -C 6 alkyl ether and C 2 -C 4 alkyl ether groups, which have 2 to 8, 6 or 4 carbon atoms, respectively.
  • a C 2 alkyl ether has the structure -CH 2 -O-CH 3 .
  • Alkoxycarbonyl groups include CpC 8 , Ci-C 6 and C]-C 4 alkoxycarbonyl groups, which have from 1 to 8, 6 or 4 carbon atoms, respectively, in the alkyl portion of the group (i.e., the carbon of the keto bridge is not included in the indicated number of carbon atoms).
  • Alkanoyloxy groups include C 2 -C 8 , C 2 -C 6 and C 2 -C 4 alkanoyloxy groups, which have from 2 to 8, 6 or 4 carbon atoms, respectively.
  • Alkanoylamino groups include C 2 -C 8 , C 2 -C 6 and C 2 -C 4 alkanoylamino groups, which have from 2 to 8, 6 or 4 carbon atoms within the alkanoyl group, respectively.
  • Alkylsulfonyl refers to groups of the formula -(SO 2 )-alkyl, in which the sulfur atom is the point of attachment. Alkylsulfonyl groups include Ci-C ⁇ alkylsulfonyl and Ci-C 4 alkylsulfonyl groups, which have from 1 to 6 or from 1 to 4 carbon atoms, respectively. Methylsulfonyl is one representative alkylsulfonyl group.
  • Ci-C 4 haloalkylsulfonyl is an alkylsulfonyl group that has from 1 to 4 carbon atoms and is substituted with at least one halogen (e.g., trifluoromethylsulfonyl) .
  • Alkylsulfonylamino refers to groups of the formula -NH-(SO 2 )-alkyl, in which the nitrogen atom is the point of attachment. Alkylsulfonylamino groups include Ci - C 6 alkylsulfonylamino and C. 1 -C 4 alkylsulfonylaniino groups, which have from 1 to 6 or 1 to 4 carbon atoms, respectively. Methylsulfonylamino is a representative alkylsulfonylamino group.
  • Aminosulfonyl refers to groups of the formula -(SO 2 )-NH 2 , in which the sulfur atom is the point of attachment.
  • the term “mono- or di-(C r C 8 alkyl)aminosulfonyl” refers to groups that satisfy the formula -(SO 2 )-NR 2 , in which the sulfur atom is the point of attachment, and in which one R is d-C 8 alkyl and the other R is hydrogen or an independently chosen Q-Cgalkyl.
  • Alkylamino refers to a secondary or tertiary amine that has the general structure -NH- alkyl or -N(alkyl)(alkyl), wherein each alkyl is selected independently from alkyl, cycloalkyl and
  • (cycloalkyl)alkyl groups include, for example, mono- and di-(Q-C 8 alkyl)amino groups, in which each C r C 8 alkyl may be the same or different, as well as mono- and di-(Q-C 8 alkyl)amino groups, in which each C r C 8 alkyl may be the same or different, as well as mono- and di-(Q-
  • Alkylaminoalkyl refers to an alkylamino group linked via an alkylene group (i.e., a group having the general structure -alkylene-NH-alkyl or -alkylene-N(alkyl)(alkyl)) in which each alkyl is selected independently from alkyl, cycloalkyl and (cycloalkyl)alkyl groups.
  • alkylene group i.e., a group having the general structure -alkylene-NH-alkyl or -alkylene-N(alkyl)(alkyl)
  • Alkylaminoalkyl groups include, for example, mono- and di-(Ci-C 8 alkyl)aminoCi-C8alkyl, mono- and di-(Ci-C 6 alkyl)aminoCi-C 6 alkyl and mono- and di-(Ci-C 6 alkyl)aminoC r C 4 alkyl.
  • "Mono- or di-(Ci-C 6 alkyl)aminoCo-C 6 alkyr' refers to a mono- or di-(Ci-C 6 alkyl)amino group linked via a single covalent bond or a C]-C 6 alkylene group.
  • alkyl as used in the terms “alkylamino” and “alkylaminoalkyl” differs from the definition of "alkyl” used for all other alkyl-containing groups, in the inclusion of cycloalkyl and (cycloalkyl)alkyl groups (e.g., (C3-C 7 cycloalkyl)Co-C 6 alkyl).
  • alkylaminoalkoxy refers to an alkylamino group linked via an alkoxy group
  • halogen refers to fluorine, chlorine, bromine or iodine.
  • haloalkyl is an alkyl group that is substituted with 1 or more independently chosen halogens (e.g., "Ci-Cghaloalkyl” groups have from 1 to 8 carbon atoms; "Ci-C 6 haloalkyl” groups have from 1 to 6 carbon atoms).
  • haloalkyl groups include, but are not limited to, mono-, di- or tri-fiuoromethyl; mono-, di- or tri-chloromethyl; mono-, di-, tri-, tetra- or penta- fluoroethyl; mono-, di-, tri-, tetra- or penta-chloroethyl; and 1,2,2,2-tetrafluoro-l-trifluoromethyl- ethyl.
  • Typical haloalkyl groups are trifluoromethyl and difluoromethyl.
  • haloalkoxy refers to a haloalkyl group as defined above attached via an oxygen bridge.
  • Ci-C 8 haloalkoxy have 1 to 8 carbon atoms.
  • a dash (“-") that is not between two letters or symbols is used to indicate a point of attachment for a substituent.
  • -CONH 2 is attached through the carbon atom.
  • a “carbocycle” or “carbocyclic group” comprises at least one ring formed entirely by carbon-carbon bonds (referred to herein as a carbocyclic ring), and does not contain a heterocycle. Unless otherwise specified, each ring within a carbocycle may be independently saturated, partially saturated or aromatic, and is optionally substituted as indicated.
  • a carbocycle generally has from 1 to 3 fused, pendant or spiro rings; carbocycles within certain embodiments have one ring or two fused rings. Typically, each ring contains from 3 to 8 ring members (i.e., C 3 -C 8 ); C 5 - C ⁇ rings are recited in certain embodiments.
  • Carbocycles comprising fused, pendant or spiro rings typically contain from 9 to 14 ring members.
  • Certain carbocycles are C 4 -Ci 0 (i.e., contain from 4 to 10 ring members, and one or two rings).
  • Certain representative carbocycles are cycloalkyl as described above.
  • Other carbocycles are aryl (i.e., contain at least one aromatic carbocyclic ring, with or without one or more additional aromatic and/or cycloalkyl rings).
  • Such aryl carbocycles include, for example, phenyl, naphthyl (e.g., 1-naphthyl and 2-naphthyl), fluorenyl, indanyl and 1,2,3,4-tetrahydro-naphthyl.
  • C 6 -Ci O arylC o -C 8 alkyl groups i.e., groups in which a 6- to 10-membered carbocyclic group comprising at least one aromatic ring is linked via a single covalent bond or a Ci-C 8 alkylene group.
  • groups include, for example, phenyl and indanyl, as well as groups in which either of the foregoing is linked via C r C 8 alkylene, preferably via C r Qalkylene.
  • Phenyl groups linked via a single covalent bond or Ci-C 6 alkylene group are designated phenylCo-C ⁇ alkyl (e.g., benzyl, 1-phenyl-ethyl, 1 -phenyl -propyl and 2-phenyl-ethyl).
  • a “heterocycle” or “heterocyclic group” has from 1 to 3 fused, pendant or spiro rings, at least one of which is a heterocyclic ring (i.e., one or more ring atoms is a heteroatom independently chosen from O, S and N, with the remaining ring atoms being carbon). Additional rings, if present, may be heterocyclic or carbocyclic. Typically, a heterocyclic ring comprises 1, 2 3 3 or 4 heteroatoms; within certain embodiments each heterocyclic ring has 1 or 2 heteroatoms per ring.
  • Each heterocyclic ring generally contains from 3 to 8 ring members (rings having from 4 or 5 to 7 ring members are recited in certain embodiments) and heterocycles comprising fused, pendant or spiro rings typically contain from 9 to 14 ring members.
  • Certain heterocycles comprise a sulfur atom as a ring member; in certain embodiments, the sulfur atom is oxidized to SO or SO 2 .
  • Heterocycles may be optionally substituted with a variety of substituents, as indicated.
  • a heterocycle may be a heterocycloalkyl group (i.e., each ring is saturated or partially saturated) or a heteroaryl group (i.e., at least one ring within the group is aromatic), such as a 5- to 10-membered heteroaryl (which may be monocyclic or bicyclic) or a 6-membered heteroaryl (e.g., pyridyl orpyrimidyl).
  • Heterocyclic groups include, for example, azepanyl, azocinyl, benzimidazolyl, benzimidazolinyl, benzisothiazolyl, benzisoxazolyl, benzofuranyl, benzothiofuranyl, benzoxazolyl, benzothiazolyl, benztetrazolyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, dihydrofuro[2,3-b]tetrahydrofuranyl, dihydroisoquinolinyl, dihydrotetrahydrofuranyl, l,4-dioxa-8- aza-spiro[4.5]decyl, dithiazinyl, furanyl, furazanyl, imidazolinyl, imidazolidinyl, imidazolyl, indazolyl, indolenyl, indolinyl
  • Certain heterocyclic groups are 4- to 12-membered, 5- to 10-membered, 3- to 7- membered, 4- to 7-membered or 5- to 7-membered groups that contain 1 heterocyclic ring or 2 fused or spiro rings, optionally substituted.
  • 4- to 10-membered heterocycloalkyl groups include, for example, piperidinyl, piperazinyl, pyrrolidinyl, azepanyl, l,4-dioxa-8-aza-spiro[4.5]dec-8-yl, morpholino, thiomorpholino and l,l-dioxo-thiomorpholin-4-yl. Such groups may be substituted as indicated.
  • Representative aromatic heterocycles are azocinyl, pyridyl, pyrimidyl, imidazolyl, tetrazolyl and 3,4-dihydro-lH-isoquinolin-2-yl.
  • a “heterocycleCo-C ⁇ alkyl” is a heterocyclic group linked via a single covalent bond or Q- Cgalkyl group.
  • a (4- to 7-membered heterocycle)C 0 -C 6 alkyl is a heterocyclic group having from 4 to 7 ring members linked via a single covalent bond or an alkyl group having from 1 to 6 carbon atoms.
  • a "(6-membered heteroaryl)C 0 -C 6 alkyl” refers to a heteroaryl group linked via a single covalent bond or Ci-C 6 alkyl group.
  • a “substituent,” as used herein, refers to a molecular moiety that is covalently bonded to an atom within a molecule of interest.
  • a ring substituent may be a moiety such as a halogen, alkyl group, haloalkyl group or other group that is covalently bonded to an atom (preferably a carbon or nitrogen atom) that is a ring member.
  • Substituents of aromatic groups are generally covalently bonded to a ring carbon atom.
  • substitution refers to replacing a hydrogen atom in a molecular structure with a substituent, such that the valence on the designated atom is not exceeded, and such that a chemically stable compound (i.e., a compound that can be isolated, characterized, and tested for biological activity) results from the substitution.
  • Groups that are "optionally substituted” are unsubstituted or are substituted by other than hydrogen at one or more available positions, typically 1, 2, 3, 4 or 5 positions, by one or more suitable groups (which may be the same or different).
  • Optional substitution is also indicated by the phrase "substituted with from 0 to X substituents," where X is the maximum number of possible substituents.
  • Certain optionally substituted groups are substituted with from 0 to 2, 3 or 4 independently selected substituents (i.e., are unsubstituted or substituted with up to the recited maximum number of substitutents).
  • Other optionally substituted groups are substituted with at least one substituent (e.g., substituted with from 1 to 2, 3 or 4 independently selected substituents).
  • VRl and “capsaicin receptor” are used interchangeably herein to refer to a type 1 vanilloid receptor. Unless otherwise specified, these terms encompass both rat and human
  • VRl receptors e.g., GenBank Accession Numbers AF327067, AJ277028 and NM_018727; sequences of certain human VRl cDNAs and the encoded amino acid sequences are provided in
  • VRl modulator also referred to herein as a “modulator,” is a compound that modulates VRl activation and/or VRl-mediated signal transduction.
  • VRl modulators specifically provided herein are compounds of Formula I and pharmaceutically acceptable salts thereof. Certain preferred VRl modulators are not vanilloids.
  • a VRl modulator may be a VRl agonist or antagonist.
  • Certain modulators bind to VRl with a K; that is less than 1 micromolar, preferably less than 500 nanomolar, 100 nanomolar, 10 nanomolar or 1 nanomolar.
  • a representative assay for determining K; at VRl is provided in Example 5, herein.
  • a modulator is considered an "antagonist” if it detectably inhibits vanilloid ligand binding to VRl and/or VRl-mediated signal transduction (using, for example, the representative assay provided in Example 6); in general, such an antagonist inhibits VRl activation with a IC S0 value of less than 1 micromolar, preferably less than 500 nanomolar, and more preferably less than 100 nanomolar, 10 nanomolar or 1 nanomolar within the assay provided in Example 6.
  • VRl antagonists include neutral antagonists and inverse agonists.
  • An "inverse agonist" of VRl is a compound that reduces the activity of VRl below its basal activity level in the absence of added vanilloid ligand.
  • Inverse agonists of VRl may also inhibit the activity of vanilloid ligand at VRl and/or binding of vanilloid ligand to VRl.
  • the basal activity of VRl, as well as the reduction in VRl activity due to the presence of VRl antagonist, may be determined from a calcium mobilization assay, such as the assay of Example 6.
  • a "neutral antagonist" of VRl is a compound that inhibits the activity of vanilloid ligand at VRl, but does not significantly change the basal activity of the receptor (i.e., within a calcium mobilization assay as described in Example 6 performed in the absence of vanilloid ligand, VRl activity is reduced by no more than 10%, preferably by no more than 5%, and more preferably by no more than 2%; most preferably, there is no detectable reduction in activity).
  • Neutral antagonists of VRl may inhibit the binding of vanilloid ligand to VRl.
  • Capsaicin receptor agonist or “VRl agonist” is a compound that elevates the activity of the receptor above the basal activity level of the receptor (i.e., enhances VRl activation and/or VRl -mediated signal transduction). Capsaicin receptor agonist activity may be identified using the representative assay provided in Example 6. In general, such an agonist has an EC50 value of less than 1 micromolar, preferably less than 500 nanomolar, and more preferably less than 100 nanomolar or 10 nanomolar within the assay provided in Example 6.
  • a “vanilloid” any compound that comprises a phenyl ring with two oxygen atoms bound to adjacent ring carbon atoms (one of which carbon atom is located para to the point of attachment of a third moiety that is bound to the phenyl ring).
  • Capsaicin is a representative vanilloid.
  • a “vanilloid ligand” is a vanilloid that binds to VRl with a K; (determined as described herein) that is no greater than 10 ⁇ M.
  • Vanilloid ligand agonists include capsaicin, olvanil, N-arachidonoyl- dopamine and resiniferatoxin (RTX).
  • Vanilloid ligand antagonists include capsazepine and iodo- resiniferatoxin.
  • a “therapeutically effective amount” is an amount that, upon administration to a patient, results in a discernible patient benefit (e.g., provides detectable relief from at least one condition being treated). Such relief may be detected using any appropriate criteria, including alleviation of one or more symptoms such as pain.
  • a therapeutically effective amount or dose generally results in a concentration of compound in a body fluid (such as blood, plasma, serum, CSF, synovial fluid, lymph, cellular interstitial fluid, tears or urine) that is sufficient to alter the binding of vanilloid ligand to VRl in vitro (using the assay provided in Example 5) and/or VRl- mediated signal transduction (using an assay provided in Example 6). It will be apparent that the discernible patient benefit may be apparent after administration of a single dose, or may become apparent following repeated administration of the therapeutically effective dose according to a predetermined regimen, depending upon the indication for which the compound is administered.
  • statically significant results varying from control at the p ⁇ 0.1 level of significance as measured using a standard parametric assay of statistical significance such as a student's T test.
  • a "patient” is any individual treated with a compound provided herein. Patients include humans, as well as other animals such as companion animals (e.g., dogs and cats) and livestock. Patients may be experiencing one or more symptoms of a condition responsive to capsaicin receptor modulation ⁇ e.g., pain, exposure to vanilloid ligand, itch, urinary incontinence, overactive bladder, respiratory disorders, cough and/or hiccup), or may be free of such symptom(s) ⁇ i.e., treatment may be prophylactic in a patient considered at risk for the development of such symptoms).
  • capsaicin receptor modulation e.g., pain, exposure to vanilloid ligand, itch, urinary incontinence, overactive bladder, respiratory disorders, cough and/or hiccup
  • the present invention provides heteroaryl substituted piperazinyl-pyridine analogues that may be used in a variety of contexts, including in the treatment of pain ⁇ e.g., neuropathic or peripheral nerve-mediated pain); exposure to capsaicin; exposure to acid, heat, light, tear gas, air pollutants (such as, for example, tobacco smoke), infectious agents (including viruses, bacteria and yeast), pepper spray or related agents; respiratory conditions such as asthma or chronic obstructive pulmonary disease; itch; urinary incontinence or overactive bladder; cough or hiccup; and/or obesity.
  • Such compounds may also be used within in vitro assays ⁇ e.g., assays for receptor activity), as probes for detection and localization of VRl and as standards in ligand binding and VRl -mediated signal transduction assays.
  • D represents a 5-membered heteroaryl with a nitrogen atom located adjacent to the point of attachment;
  • D, E and G are independently O, S, N, NR la or CRj 3 ;
  • Each Ri a is independently chosen from:
  • compounds provided herein satisfy one or more of Formulas IIa — lid, in which variables are as indicated for Formula II, except as defined below:
  • R la in the above groups is not hydrogen.
  • R ia is hydrogen, halogen, amino, cyano, C r C 6 alkyl, Ci- C 6 haloalkyl, Ci-C 6 alkoxy, C]-C 6 alkylsulfonyl, or mono- or di-(Ci-C 6 alkyl)aminosulfonyl.
  • Representative Ri a groups include, for example, hydrogen, halogen, nitro, cyano, methyl, and trifluoromethyl.
  • Ar 2 is optionally substituted phenyl, pyridyl ⁇ i.e., 2-pyridyl, 3-pyridyl or 4- ⁇ yridyl) or pyrimidyl.
  • Ar 2 is a 9- to 12- membered bicyclic aryl or heteroaryl group that is optionally substituted as described above.
  • Ar 2 is substituted with from 0 to 3 or from 1 to 3 substituents independently chosen from R 2 as described above.
  • Ar 2 has at least one substituent (R 2 ) and each R 2 is independently chosen from amino, cyano, halogen, -SO 2 NH 2 , Ci-C 4 alkyl, Q-C 4 haloalkyl, Q- C 4 hydroxyalkyl, Q-C 4 alkoxy, Q-Qalkylthio, Ci-C 4 haloalkoxy 5 mono- and di-(Q- C 4 alkyl)aminoCo-C 4 alkyl, C 2 -C 4 alkanoyl, C 1 -C 4 alkoxycarbonyl, d-C 4 alkylsulfonyl Q- C 4 haloalkylsulfonyl and mono- and di-(Ci-Qalkyl)aminosulfonyl.
  • the substituents of Ar 2 are independently chosen from amino, cyano, halogen, Q-C 4 alkyl, Q- Qhaloalkyl, Q-C 4 alkoxy, Ci-C 4 haloalkoxy, Ci-Qalkylthio and mono- and di-(Q- C 4 alkyl)aminoCo-Qalkyl.
  • Ar 2 is substituted meta and/or para to the point of attachment, wherein the point of attachment refers to the attachment to the core ring.
  • Ar 2 is phenyl
  • the phenyl is mono-substituted at the 3 -position, mono-substituted at the 4-position, or di-substituted and the 3- and 4-positions.
  • Preferred Ar 2 groups include phenyl, pyridyl and pyrimidyl, each of which is substituted with from 0 to 3 or from 1 to 3 substituents as described herein.
  • one R 2 is taken together with an adjacent R 2 to form a fused carbocycle or heterocycle.
  • Representative such groups include, for example, the following bicyclic groups, optionally substituted as described herein:
  • each R 2a is independently chosen from amino, cyano, halogen, -SO 2 NH 2 , -COOH, Ci-C 4 alkyl, C 1 - C 4 haloalkyl, Ci-C 4 hydroxyalkyl, Ci-C 4 alkoxy, C r C 4 alkylthio, C r C 4 haloalkoxy, mono- and di- (Ci-C 4 alkyl)aminoC 0 -C 4 alkyl, C 2 -C 4 alkanoyl, C]-C 4 alkoxycarbonyl, Ci-C 4 alkylsulfonyl, Q- C 4 haloalkylsulfonyl and mono- and di-(Ci-C 4 alkyl)sulfonamido.
  • A is CH or CR 2a , and each R 2a is independently chosen from cyano, halogen, Q- ⁇ alkyl, C r C 4 haloalkyl, Ci-C 4 alkoxy and mono- and di-(C r C 4 alkyl)aminoC 0 -C 2 alkyl.
  • R 2a is independently chosen from cyano, halogen, Q- ⁇ alkyl, C r C 4 haloalkyl, Ci-C 4 alkoxy and mono- and di-(C r C 4 alkyl)aminoC 0 -C 2 alkyl.
  • at least one of A, B, E and T is N.
  • Certain Ar 2 groups have the formula:
  • L is defined as Co-C 6 alkylene or Ci-C 6 alkylene that is taken together with R 5 , R 6 or R 7 to form a 4- to 7-membered heterocycle. In any heterocycle so formed, at least one carbon atom present in L is also a ring atom, and is covalently bonded to a component atom OfR 5 , R 6 or R 7 .
  • the resulting heterocycle may be a heterocycloalkyl group (e.g., tetrahydrofuranyl, morpholinyl, piperidinyl or piperazinyl) or a heteroaryl group, such as pyridyl, pyrimidyl or tetrahydrofuranyl.
  • R 3 groups comprising such a heterocycle include, for example:
  • R 3 in certain embodiments of the above Formulas, is a group of the formula:
  • L is C 0 -C 3 alkyl; and R 5 and R 6 are:
  • R 3 groups include, for example, mono- and di-(Ci-C 4 alkyl)ammo groups that are
  • R 3 groups include phenyl and 4- to 7-membered heterocycles, each of which is
  • R 3 groups include azetidinyl, pyrrolidinyl, morpholinyl, thiomorpholinyl, piperidinyl, piperazinyl, tetrahydropyridyl and azepanyl, each of which is substituted with from O to 4 substituents independently chosen from: (a) halogen, hydroxy, amino, oxo, aminocarbonyl, -SO 2 NH 2 and -
  • R 3 groups include the heterocycles:
  • R 3 groups are phenyl, pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, imidazolyl, thienyl, oxazolyl or tetrahydrofuranyl, each of which is substituted with from 0 to 4 substituents independently chosen from halogen, hydroxy, amino, aminocarbonyl, -SO 2 NH 2 , -COOH, Cj- C 4 alkyl, C 5 -C 7 cycloalkyl, C 2 -C 4 alkyl ether, Ci-C 4 alkoxy, C 2 -C 4 alkanoyl, Ci-C 4 haloalkyl and mono- and di-(Ci-C 4 alkyl)amino.
  • R 3 is not -NH 2 . In other words, if L is a single covalent bond then at least one of R 5 and R 6 is not hydrogen.
  • R 3 is 7 .
  • L is C 0 -C 3 alkyl; and R 7 is Ci-C 6 alkyl, C 2 -C 6 alkenyl, (C5-C 7 cycloalkyl)Co-C 4 alkyl, C 2 -C 4 alkanoyl, phenylC 0 -C 6 alkyl or (6-membered heteroaryl)C 0 -C 4 alkyl, each of which is substituted with from O to 4 substituents independently chosen from halogen, hydroxy, amino, aminocarbonyl, -SO 2 NH 2 , -COOH, Q- C 4 alkyl, C 5 -C 7 cycloalkyl, C 2 -C 4 alkyl ether, C r C 4 alkoxy, C 2 -C 4 alkanoyl, Ci-C 4 haloalkyl and mono- and di-(C 1 -C 4 alkyl)amino.
  • R 3 groups include
  • R 3 is a hydrogen, Ci-C 6 alkyl or a halogen.
  • R 3 is Ci-C 4 alkyl, C 3 -Cycycloalkyl or Q- C 4 haloalkyl, each of which is substituted with from O to 4 substituents independently chosen from halogen, hydroxy, cyano, oxo, aminocarbonyl, -SO 2 NH 2 , -COOH, C 3 -C 7 cycloalkyl, phenyl and 4- to 7-membered heterocycle.
  • R 4 in certain compounds provided herein, represents zero substituents or one methyl, ethyl or oxo group, preferably located at the 2-position of the piperazine.
  • the carbon to which a methyl or ethyl group is attached is chiral in certain embodiments.
  • the group: is , , or in certain compounds.
  • R 4 represents a single oxo substituent.
  • X, Y and Z are independently CR x or N, such that at least one of X, Y and Z is N.
  • each R x is independently chosen from hydrogen and methyl, or each R x is hydrogen.
  • Z is N (e.g., X and Y are CH).
  • X is N (e.g., Y and Z are CH).
  • Z and X are N,
  • X and Y are N or Z and Y are N. In further compounds, X, Y and Z are each N.
  • D, E and G are independently O, S, N, NR la or CR la ;
  • Each Ru is independently chosen from:
  • Ar 2 is phenyl, pyridyl or pyrimidyl, each of which is substituted with from 0 to 3 substituents independently chosen from amino, cyano, halogen, C]-C 4 alkyl, Ci-C 4 haloalkyl, Ci-C 4 alkoxy, C]-C 4 haloalkoxy, Ci-C 4 alkylthio, and mono- and di-(Ci-C 4 alkyl)aminoC 0 -C 4 alkyl; and R 4 represents 0 substituents or one methyl substituent.
  • Representative compounds provided herein include, but are not limited to, those specifically described in Examples 1-3.
  • heteroaryl substituted piperazinyl-pyridine analogues detectably alter (modulate) VRl activity, as determined using an in vitro VRl functional assay such as a calcium mobilization assay.
  • a VRl ligand binding assay may be used.
  • References herein to a "VRl ligand binding assay” are intended to refer to a standard in vitro receptor binding assay such as that provided in Example 5, and a "calcium mobilization assay” (also referred to herein as a “signal transduction assay”) may be performed as described in Example 6.
  • a competition assay may be performed in which a VRl preparation is incubated with labeled (e.g., 125 I or 3 H) compound that binds to VRl (e.g., a capsaicin receptor agonist such as RTX) and unlabeled test compound.
  • labeled e.g., 125 I or 3 H
  • VRl e.g., a capsaicin receptor agonist such as RTX
  • the VRl used is preferably mammalian VRl, more preferably human or rat VRl.
  • the receptor may be recombinantly expressed or naturally expressed.
  • the VRl preparation may be, for example, a membrane preparation from HEK293 or CHO cells that recombinantly express human VRl.
  • Incubation with a compound that detectably modulates vanilloid ligand binding to VRl results in a decrease or increase in the amount of label bound to the VRl preparation, relative to the amount of label bound in the absence of the compound. This decrease or increase may be used to determine the Kj at VRl as described herein. In general, compounds that decrease the amount of label bound to the VRl preparation within such an assay are preferred.
  • Certain VRl modulators provided herein detectably modulate VRl activity at nanomolar (i.e., submicromolar) concentrations, at subnanomolar concentrations, or at concentrations below 100 picomolar, 20 picomolar, 10 picomolar or 5 picomolar.
  • IC 50 values for such compounds may be determined using a standard in vitro VRl- mediated calcium mobilization assay, as provided in Example 6. Briefly, cells expressing capsaicin receptor are contacted with a compound of interest and with an indicator of intracellular calcium concentration (e.g., a membrane permeable calcium sensitivity dye such as Fluo-3 or Fura-2 (Molecular Probes, Eugene, OR), each of which produce a fluorescent signal when bound to Ca +4 ). Such contact is preferably carried out by one or more incubations of the cells in buffer or culture medium comprising either or both of the compound and the indicator in solution.
  • an indicator of intracellular calcium concentration e.g., a membrane permeable calcium sensitivity dye such as Fluo-3 or Fura-2 (Molecular Probes, Eugene, OR)
  • a vanilloid receptor agonist e.g., capsaicin, RTX or olvanil
  • a fluorescence response is measured.
  • agonist-contacted cells are contacted with a compound that is a VRl antagonist the fluorescence response is generally reduced by at least 20%, preferably at least 50% and more preferably at least 80%, as compared to cells that are contacted with the agonist in the absence of test compound.
  • the IC 50 for VRl antagonists provided herein is preferably less than 1 micromolar, less than 100 nM, less than 10 nM or less than 1 nM.
  • VRl antagonists provided herein exhibit no detectable agonist activity an in vitro assay of capsaicin receptor agonism at a concentration of compound equal to the IC5 0 .
  • Certain such antagonists exhibit no detectable agonist activity an in vitro assay of capsaicin receptor agonism at a concentration of compound that is 100-fold higher than the IC 50 .
  • compounds that are capsaicin receptor agonists are preferred. Capsaicin receptor agonist activity may generally be determined as described in Example 6.
  • the fluorescence response is generally increased by an amount that is at least 30% of the increase observed when cells are contacted with 100 nM capsaicin.
  • the EC 50 for VRl agonists provided herein is preferably less than 1 micromolar, less than 100 nM or less than 10 nM.
  • VRl modulating activity may also, or alternatively, be assessed using a cultured dorsal root ganglion assay as provided in Example 7 and/or an in vivo pain relief assay as provided in Example 8.
  • VRl modulators provided herein preferably have a statistically significant specific effect on VRl activity within one or more functional assays provided herein.
  • VRl modulators provided herein do not substantially modulate ligand binding to other cell surface receptors, such as EGF receptor tyrosine kinase or the nicotinic acetylcholine receptor.
  • such modulators do not substantially inhibit activity of a cell surface receptor such as the human epidermal growth factor (EGF) receptor tyrosine kinase or the nicotinic acetylcholine receptor (e.g., the IC 50 or IC 40 at such a receptor is preferably greater than 1 micromolar, and most preferably greater than 10 micromolar).
  • EGF human epidermal growth factor
  • a modulator does not detectably inhibit EGF receptor activity or nicotinic acetylcholine receptor activity at a concentration of 0.5 micromolar, 1 micromolar or more preferably 10 micromolar.
  • Assays for determining cell surface receptor activity are commercially available, and include the tyrosine kinase assay kits available from Panvera (Madison, WI).
  • preferred VRl modulators are non-sedating.
  • a dose of VRl modulator that is twice the minimum dose sufficient to provide analgesia in an animal model for determining pain relief causes only transient (i.e., lasting for no more than 1 A the time that pain relief lasts) or preferably no statistically significant sedation in an animal model assay of sedation (using the method described by Fitzgerald et al. (1988) Toxicology 49(2-3):433-9).
  • a dose that is five times the minimum dose sufficient to provide analgesia does not produce statistically significant sedation.
  • a VRl modulator provided herein does not produce sedation at intravenous doses of less than 25 mg/kg (preferably less than 10 mg/kg) or at oral doses of less than 140 mg/kg (preferably less than 50 mg/kg, more preferably less than 30 mg/kg).
  • compounds provided herein may be evaluated for certain pharmacological properties including, but not limited to, oral bioavailability (preferred compounds are orally bioavailable to an extent allowing for therapeutically effective concentrations of the compound to be achieved at oral doses of less than 140 mg/kg, preferably less than 50 mg/kg, more preferably less than 30 mg/kg, even more preferably less than 10 mg/kg, still more preferably less than 1 mg/kg and most preferably less than 0.1 mg/kg), toxicity (a preferred compound is nontoxic when a therapeutically effective amount is administered to a subject), side effects (a preferred compound produces side effects comparable to placebo when a therapeutically effective amount of the compound is administered to a subject), serum protein binding and in vitro and in vivo half-life (a preferred compound exhibits an in vivo half-life allowing for Q.I.D.
  • assays used to predict bioavailability include transport across human intestinal cell monolayers, including Caco-2 cell monolayers.
  • Penetration of the blood brain barrier of a compound in humans may be predicted from the brain levels of the compound in laboratory animals given the compound (e.g., intravenously).
  • Serum protein binding may be predicted from albumin binding assays.
  • nontoxic shall be understood in a relative sense and is intended to refer to any substance that has been approved by the United States Food and Drug Administration (“FDA”) for administration to mammals (preferably humans) or, in keeping with established criteria, is susceptible to approval by the FDA for administration to mammals (preferably humans).
  • FDA United States Food and Drug Administration
  • a highly preferred nontoxic compound generally satisfies one or more of the following criteria: (1) does not substantially inhibit cellular ATP production; (2) does not significantly prolong heart QT intervals; (3) does not cause substantial liver enlargement, or (4) does not cause substantial release of liver enzymes.
  • a compound that does not substantially inhibit cellular ATP production is a compound that satisfies the criteria set forth in Example 8 of published U.S. Application Number 2005/0070547.
  • cells treated as described therein with 100 ⁇ M of such a compound exhibit ATP levels that are at least 50% of the ATP levels detected in untreated cells.
  • such cells exhibit ATP levels that are at least 80% of the ATP levels detected in untreated cells.
  • a compound that does not significantly prolong heart QT intervals is a compound that does not result in a statistically significant prolongation of heart QT intervals (as determined by electrocardiography) in guinea pigs, minipigs or dogs upon administration of a dose that yields a serum concentration equal to the EC 50 or IC 5Q for the compound.
  • a dose of 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 40 or 50 mg/kg administered parenterally or orally does not result in a statistically significant prolongation of heart QT intervals.
  • a compound does not cause substantial liver enlargement if daily treatment of laboratory rodents ⁇ e.g., mice or rats) for 5-10 days with a dose that yields a serum concentration equal to the EC 50 or IC 50 for the compound results in an increase in liver to body weight ratio that is no more than 100% over matched controls. In more highly preferred embodiments, such doses do not cause liver enlargement of more than 75% or 50% over matched controls. If non-rodent mammals (e.g., dogs) are used, such doses should not result in an increase of liver to body weight ratio of more than 50%, preferably not more than 25%, and more preferably not more than 10% over matched untreated controls. Preferred doses within such assays include 0.01, 0.05. 0.1, 0.5, 1, 5, 10, 40 or 50 mg/kg administered parenterally or orally.
  • a compound does not promote substantial release of liver enzymes if administration of twice the minimum dose that yields a serum concentration equal to the EC 50 or IC 50 at VRl for the compound does not elevate serum levels of ALT, LDH or AST in laboratory animals ⁇ e.g., rodents) by more than 100% over matched mock-treated controls. In more highly preferred embodiments, such doses do not elevate such serum levels by more than 75% or 50% over matched controls.
  • a compound does not promote substantial release of liver enzymes if, in an in vitro hepatocyte assay, concentrations (in culture media or other such solutions that are contacted and incubated with hepatocytes in vitro) that are equal to the EC 50 or IC 50 for the compound do not cause detectable release of any of such liver enzymes into culture medium above baseline levels seen in media from matched mock-treated control cells. In more highly preferred embodiments, there is no detectable release of any of such liver enzymes into culture medium above baseline levels when such compound concentrations are five-fold, and preferably ten-fold the EC 5O or IC 50 for the compound.
  • certain preferred compounds do not inhibit or induce microsomal cytochrome P450 enzyme activities, such as CYP1A2 activity, CYP2A6 activity, CYP2C9 activity, CYP2C19 activity, CYP2D6 activity, CYP2E1 activity or CYP3A4 activity at a concentration equal to the EC 50 or IC 50 at VRl for the compound.
  • microsomal cytochrome P450 enzyme activities such as CYP1A2 activity, CYP2A6 activity, CYP2C9 activity, CYP2C19 activity, CYP2D6 activity, CYP2E1 activity or CYP3A4 activity at a concentration equal to the EC 50 or IC 50 at VRl for the compound.
  • Certain preferred compounds are not clastogenic ⁇ e.g., as determined using a mouse erythrocyte precursor cell micronucleus assay, an Ames micronucleus assay, a spiral micronucleus assay or the like) at a concentration equal the EC 50 or IC 50 for the compound.
  • certain preferred compounds do not induce sister chromatid exchange ⁇ e.g., in Chinese hamster ovary cells) at such concentrations.
  • VRl modulators provided herein may be isotopically-labeled or radiolabeled.
  • compounds may have one or more atoms replaced by an atom of the same element having an atomic mass or mass number different from the atomic mass or mass number usually found in nature.
  • isotopes that can be present in the compounds provided herein include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine, such as 2 H, 3 H, 11 C, 13 C, 14 C, 15 N 5 18 O, 17 0, 31 P, 32 P, 35 S, 18 F and 36 Cl.
  • substitution with heavy isotopes such as deuterium (i.e., 2 H) can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances.
  • Heteroaryl substituted piperazinyl-pyridine analogues may generally be prepared using standard synthetic methods. Starting materials are commercially available from suppliers such as Sigma-Aldrich Corp. (St. Louis, MO), or may be synthesized from commercially available precursors using established protocols. By way of example, a synthetic route similar to that shown in any of the following Schemes may be used, together with synthetic methods known in the art of synthetic organic chemistry, or variations thereon as appreciated by those skilled in the art. Each variable in the following schemes refers to any group consistent with the description of the compounds provided herein.
  • catalyst refers to a suitable transition metal catalyst such as, but not limited to, tetrakis(triphenylphosphine)palladium(0) or palladium(II) acetate.
  • the catalytic systems may include ligands such as, but not limited to, 2- (Dicyclohexylphosphino)biphenyl and tri-ter/-butylphosphine, and may also include a base such as K 3 PO 4 , Na 2 CO 3 or sodium or potassium tert-hutoxide.
  • Transition metal-catalyzed reactions can be carried out at ambient or elevated temperatures using various inert solvents including, but not limited to, toluene, dioxane, DMF, N-methylpyrrolidinone, ethyleneglycol, dimethyl ether, diglyme and acetonitrile.
  • inert solvents including, but not limited to, toluene, dioxane, DMF, N-methylpyrrolidinone, ethyleneglycol, dimethyl ether, diglyme and acetonitrile.
  • transition metal-catalyzed (hetero)aryl/aryl coupling reactions can be used to prepare the compounds encompassed in general structures 5C (Scheme 5), 6-D and 6-E (Scheme 6) and 7-F (Scheme 7).
  • reagent/catalyst pairs include aryl boronic acid/palladium(O) (Suzuki reaction; Miyaura and Suzuki (1995) Chemical Reviews 95:2457) and aryl trialkylstannane/palladium(O) (Stille reaction; T. N. Mitchell, (1992) Synthesis P:803-815), arylzinc/palladium(O) and aryl Grignard/nickel(II).
  • metal-catalyzed (hetero)aryl/amine coupling reactions Buchwald-Hartwig cross-coupling reaction; J. F. Hartwig, Angew. Chem. Int. Ed.
  • PhNEt 2 diethyl-phenyl-amine, also referred to as ⁇ , ⁇ -diethylaniline
  • a compound provided herein may contain one or more asymmetric carbon atoms, so that the compound can exist in different stereoisomeric forms.
  • Such forms can be, for example, racemates or optically active forms.
  • All stereoisomers are encompassed by the present invention. Nonetheless, it may be desirable to obtain single enantiomers (i.e., optically active forms).
  • Standard methods for preparing single enantiomers include asymmetric synthesis and resolution of the racemates. Resolution of the racemates can be accomplished, for example, by conventional methods such as crystallization in the presence of a resolving agent, or chromatography using, for example a chiral HPLC column.
  • Compounds may be radiolabeled by carrying out their synthesis using precursors comprising at least one atom that is a radioisotope. Each radioisotope is preferably carbon (e.g.,
  • Tritium labeled compounds may also be prepared catalytically via platinum-catalyzed exchange in tritiated acetic acid, acid- catalyzed exchange in tritiated trifluoroacetic acid, or heterogeneous-catalyzed exchange with tritium gas using the compound as substrate.
  • certain precursors may be subjected to tritium-halogen exchange with tritium gas, tritium gas reduction of unsaturated bonds, or reduction using sodium borotritide, as appropriate.
  • Preparation of radiolabeled compounds may be conveniently performed by a radioisotope supplier specializing in custom synthesis of radiolabeled probe compounds.
  • compositions comprising one or more compounds provided herein, together with at least one physiologically acceptable carrier or excipient.
  • Pharmaceutical compositions may comprise, for example, one or more of water, buffers (e.g., neutral buffered saline or phosphate buffered saline), ethanol, mineral oil, vegetable oil, dimethylsulfoxide, carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, adjuvants, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione and/or preservatives.
  • other active ingredients may (but need not) be included in the pharmaceutical compositions provided herein.
  • compositions may be formulated for any appropriate manner of administration, including, for example, topical, oral, nasal, rectal or parenteral administration.
  • parenteral as used herein includes subcutaneous, intradermal, intravascular (e.g., intravenous), intramuscular, spinal, intracranial, intrathecal and intraperitoneal injection, as well as any similar injection or infusion technique.
  • compositions suitable for oral use are preferred. Such compositions include, for example, tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs.
  • pharmaceutical compositions may be formulated as a lyophilizate.
  • Formulation for topical administration may be preferred for certain conditions (e.g., in the treatment of skin conditions such as burns or itch).
  • Formulation for direct administration into the bladder may be preferred for treatment of urinary incontinence and overactive bladder.
  • Compositions intended for oral use may further comprise one or more components such as sweetening agents, flavoring agents, coloring agents and/or preserving agents in order to provide appealing and palatable preparations.
  • Tablets contain the active ingredient in admixture with physiologically acceptable excipients that are suitable for the manufacture of tablets.
  • excipients include, for example, inert diluents (e.g., calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate), granulating and disintegrating agents (e.g., corn starch or alginic acid), binding agents (e.g., starch, gelatin or acacia) and lubricating agents (e.g., magnesium stearate, stearic acid or talc).
  • inert diluents e.g., calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate
  • granulating and disintegrating agents e.g., corn starch or
  • the tablets may be uncoated or they may be coated by known techniques.
  • Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent (e.g., calcium carbonate, calcium phosphate or kaolin), or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium (e.g., peanut oil, liquid paraffin or olive oil).
  • an inert solid diluent e.g., calcium carbonate, calcium phosphate or kaolin
  • an oil medium e.g., peanut oil, liquid paraffin or olive oil
  • Aqueous suspensions contain the active material(s) in admixture with suitable excipients, such as suspending agents (e.g., sodium carboxymethylcellulose, methylcellulose, hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia); and dispersing or wetting agents (e.g., naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with fatty acids such as polyoxyethylene stearate, condensation products of ethylene oxide with long chain aliphatic alcohols such as heptadecaethyleneoxycetanol, condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides such as polyethylene sorbitan monooleate).
  • suspending agents e.g., sodium carb
  • Aqueous suspensions may also comprise one or more preservatives, such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and/or one or more sweetening agents, such as sucrose or saccharin.
  • Oily suspensions may be formulated by suspending the active ingredient(s) in a vegetable oil (e.g., arachis oil, olive oil, sesame oil or coconut oil) or in a mineral oil such as liquid paraffin.
  • the oily suspensions may contain a thickening agent such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and/or flavoring agents may be added to provide palatable oral preparations.
  • Such suspensions may be preserved by the addition of an antioxidant such as ascorbic acid.
  • Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent and one or more preservatives.
  • a dispersing or wetting agent e.g., a suspending agent
  • Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, such as sweetening, flavoring and coloring agents, may also be present.
  • compositions may also be formulated as oil-in-water emulsions.
  • the oily phase may be a vegetable oil (e.g., olive oil or arachis oil), a mineral oil (e.g., liquid paraffin) or a mixture thereof.
  • Suitable emulsifying agents include naturally-occurring gums (e.g., gum acacia or gum tragacanth), naturally-occurring phosphatides (e.g., soy bean lecithin, and esters or partial esters derived from fatty acids and hexitol), anhydrides (e.g., sorbitan monoleate) and condensation products of partial esters derived from fatty acids and hexitol with ethylene oxide (e.g., polyoxyethylene sorbitan monoleate).
  • An emulsion may also comprise one or more sweetening and/or flavoring agents.
  • Syrups and elixirs may be formulated with sweetening agents, such as glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also comprise one or more demulcents, preservatives, flavoring agents and/or coloring agents.
  • sweetening agents such as glycerol, propylene glycol, sorbitol or sucrose.
  • Such formulations may also comprise one or more demulcents, preservatives, flavoring agents and/or coloring agents.
  • Formulations for topical administration typically comprise a topical vehicle combined with active agent(s), with or without additional optional components.
  • Suitable topical vehicles and additional components are well known in the art, and it will be apparent that the choice of a vehicle will depend on the particular physical form and mode of delivery.
  • Topical vehicles include water; organic solvents such as alcohols (e.g., ethanol or isopropyl alcohol) or glycerin; glycols ⁇ e.g., butylene, isoprene or propylene glycol); aliphatic alcohols (e.g., lanolin); mixtures of water and organic solvents and mixtures of organic solvents such as alcohol and glycerin; lipid-based materials such as fatty acids, acylglycerols (including oils, such as mineral oil, and fats of natural or synthetic origin), phosphoglycerides, sphingolipids and waxes; protein-based materials such as collagen and gelatin; silicone-based materials (both non-volatile and volatile); and hydrocarbon- based materials such as microsponges and polymer matrices.
  • organic solvents such as alcohols (e.g., ethanol or isopropyl alcohol) or glycerin
  • a composition may further include one or more components adapted to improve the stability or effectiveness of the applied formulation, such as stabilizing agents, suspending agents, emulsifying agents, viscosity adjusters, gelling agents, preservatives, antioxidants, skin penetration enhancers, moisturizers and sustained release materials.
  • stabilizing agents such as stabilizing agents, suspending agents, emulsifying agents, viscosity adjusters, gelling agents, preservatives, antioxidants, skin penetration enhancers, moisturizers and sustained release materials.
  • stabilizing agents such as hydroxymethylcellulose or gelatin-microcapsules, liposomes, albumin microspheres, microemulsions, nanoparticles or nanocapsules.
  • a topical formulation may be prepared in any of a variety of physical forms including, for example, solids, pastes, creams, foams, lotions, gels, powders, aqueous liquids and emulsions.
  • the physical appearance and viscosity of such pharmaceutically acceptable forms can be governed by the presence and amount of emulsifier(s) and viscosity adjuster(s) present in the formulation.
  • Solids are generally firm and non-pourable and commonly are formulated as bars or sticks, or in particulate form; solids can be opaque or transparent, and optionally can contain solvents, emulsifiers, moisturizers, emollients, fragrances, dyes/colorants, preservatives and other active ingredients that increase or enhance the efficacy of the final product.
  • Creams and lotions are often similar to one another, differing mainly in their viscosity; both lotions and creams may be opaque, translucent or clear and often contain emulsifiers, solvents, and viscosity adjusting agents, as well as moisturizers, emollients, fragrances, dyes/colorants, preservatives and other active ingredients that increase or enhance the efficacy of the final product.
  • Gels can be prepared with a range of viscosities, from thick or high viscosity to thin or low viscosity.
  • These formulations may also contain solvents, emulsifiers, moisturizers, emollients, fragrances, dyes/colorants, preservatives and other active ingredients that increase or enhance the efficacy of the final product.
  • Liquids are thinner than creams, lotions, or gels and often do not contain emulsifiers.
  • Liquid topical products often contain solvents, emulsifiers, moisturizers, emollients, fragrances, dyes/colorants, preservatives and other active ingredients that increase or enhance the efficacy of the final product.
  • Suitable emulsifiers for use in topical formulations include, but are not limited to, ionic emulsifiers, cetearyl alcohol, non-ionic emulsifiers like polyoxyethylene oleyl ether, PEG-40 stearate, ceteareth-12, ceteareth-20, ceteareth-30, ceteareth alcohol, PEG-100 stearate and glyceryl stearate.
  • Suitable viscosity adjusting agents include, but are not limited to, protective colloids or non-ionic gums such as hydroxyethylcellulose, xanthan gum, magnesium aluminum silicate, silica, microcrystalline wax, beeswax, paraffin, and cetyl palmitate.
  • a gel composition may be formed by the addition of a gelling agent such as chitosan, methyl cellulose, ethyl cellulose, polyvinyl alcohol, polyquaterniums, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carbomer or ammoniated glycyrrhizinate.
  • a gelling agent such as chitosan, methyl cellulose, ethyl cellulose, polyvinyl alcohol, polyquaterniums, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carbomer or ammoniated glycyrrhizinate.
  • Suitable surfactants include, but are not limited to, nonionic, amphoteric, ionic and anionic surfactants.
  • dimethicone copolyol polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, lauramide DEA, cocamide DEA, and cocamide MEA, oleyl betaine, cocamidopropyl phosphatidyl PG-dimonium chloride, and ammonium laureth sulfate may be used within topical formulations.
  • Suitable preservatives include, but are not limited to, antimicrobials such as methylparaben, propylparaben, sorbic acid, benzoic acid, and formaldehyde, as well as physical stabilizers and antioxidants such as vitamin E, sodium ascorbate/ascorbic acid and propyl gallate.
  • Suitable moisturizers include, but are not limited to, lactic acid and other hydroxy acids and their salts, glycerin, propylene glycol, and butylene glycol.
  • Suitable emollients include lanolin alcohol, lanolin, lanolin derivatives, cholesterol, petrolatum, isostearyl neopentanoate and mineral oils.
  • Suitable fragrances and colors include, but are not limited to, FD&C Red No. 40 and FD&C Yellow No. 5.
  • Suitable additional ingredients include, but are not limited to, abrasives, absorbents, anti-caking agents, anti-foaming agents, antistatic agents, astringents (e.g., witch hazel, alcohol and herbal extracts such as chamomile extract), binders/excipients, buffering agents, chelating agents, film forming agents, conditioning agents, propellants, opacifying agents, pH adjusters and protectants.
  • An example of a suitable topical vehicle for formulation of a gel is: hydroxypropylcellulose (2.1%); 70/30 isopropyl alcohol/water (90.9%); propylene glycol (5.1%); and Polysorbate 80 (1.9%).
  • An example of a suitable topical vehicle for formulation as a foam is: cetyl alcohol (1.1%); stearyl alcohol (0.5%; Quaternium 52 (1.0%); propylene glycol (2.0%); Ethanol 95 PGF3 (61.05%); deionized water (30.05%); P75 hydrocarbon propellant (4.30%). All percents are by weight.
  • Typical modes of delivery for topical compositions include application using the fingers; application using a physical applicator such as a cloth, tissue, swab, stick or brush; spraying (including mist, aerosol or foam spraying); dropper application; sprinkling; soaking; and rinsing.
  • a pharmaceutical composition may be prepared as a sterile injectible aqueous or oleaginous suspension.
  • the compound(s) provided herein, depending on the vehicle and concentration used, can either be suspended or dissolved in the vehicle.
  • Such a composition may be formulated according to the known art using suitable dispersing, wetting agents and/or suspending agents such as those mentioned above.
  • suitable dispersing, wetting agents and/or suspending agents such as those mentioned above.
  • suitable dispersing, wetting agents and/or suspending agents such as those mentioned above.
  • suitable dispersing, wetting agents and/or suspending agents such as those mentioned above.
  • suitable dispersing, wetting agents and/or suspending agents such as those mentioned above.
  • suitable dispersing, wetting agents and/or suspending agents such as those mentioned above.
  • suitable dispersing, wetting agents and/or suspending agents such as those mentioned above.
  • suitable vehicles and solvents that may be employed are water, 1,3-butanediol, Ringer's solution
  • compositions may also be formulated as suppositories (e.g., for rectal administration). Such compositions can be prepared by mixing the drug with a suitable non- irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Suitable excipients include, for example, cocoa butter and polyethylene glycols.
  • compositions for inhalation typically can be provided in the form of a solution, suspension or emulsion that can be administered as a dry powder or in the form of an aerosol using a conventional propellant (e.g., dichlorodifluoromethane or trichlorofluoromethane).
  • a conventional propellant e.g., dichlorodifluoromethane or trichlorofluoromethane.
  • compositions may be formulated as sustained release or controlled-release formulations (i.e., a formulation such as a capsule that effects a slow release of active ingredient(s) following administration).
  • sustained release or controlled-release formulations i.e., a formulation such as a capsule that effects a slow release of active ingredient(s) following administration.
  • Such formulations may generally be prepared using well known technology and administered by, for example, oral, rectal or subcutaneous implantation, or by implantation at the desired target site.
  • the formulation provides a relatively constant level of release of active ingredient(s); the release profile can be varied using methods well known in the art, including (a) by varying the thickness or composition of the coating, (b) by altering the amount or manner of addition of plasticizer in the coating, (c) by including additional ingredients, such as release-modifying agents, (d) by altering the composition, particle size or particle shape of the matrix, and (e) by providing one or more passageways through the coating.
  • the amount of modulator contained within a sustained release formulation depends upon, for example, the method of administration (e.g., the site of implantation), the rate and expected duration of release and the nature of the condition to be treated or prevented.
  • a sustained and/or controlled release formulation comprises a matrix and/or coating that delays disintegration and absorption in the gastrointestinal tract (or implantation site) and thereby provides a delayed action or a sustained action over a longer period.
  • a time delay material such as glyceryl monosterate or glyceryl distearate may be employed.
  • Coatings that regulate release of the modulator include pH-dependent coatings (which may be used to release modulator in the stomach, and enteric coatings (which may be used to release modulator further along the gastrointestinal tract). pH dependent coatings include, for example, shellac, cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropylmethylcellulose phthalate, methacrylic acid ester copolymers and zein.
  • a compound provided herein may be conveniently added to food or drinking water (e.g., for administration to non-human animals including companion animals (such as dogs and cats) and livestock).
  • Animal feed and drinking water compositions may be formulated so that the animal takes in an appropriate quantity of the composition along with its diet. It may also be convenient to present the composition as a premix for addition to feed or drinking water.
  • Compounds are generally administered in a therapeutically effective amount.
  • Preferred systemic doses are no higher than 50 mg per kilogram of body weight per day (e.g., ranging from about 0.001 mg to about 50 mg per kilogram of body weight per day), with oral doses generally being about 5-20 fold higher than intravenous doses (e.g., ranging from 0.01 to 40 mg per kilogram of body weight per day).
  • the amount of active ingredient that may be combined with the carrier materials to produce a single dosage unit will vary depending, for example, upon the patient being treated, the particular mode of administration and any other co-administered drugs. Dosage units generally contain between from about 10 ⁇ g to about 500 mg of active ingredient. Optimal dosages may be established using routine testing, and procedures that are well known in the art.
  • compositions may be packaged for treating conditions responsive to VRl modulation (e.g., treatment of exposure to vanilloid ligand or other irritant, pain, itch, obesity or urinary incontinence).
  • Packaged pharmaceutical compositions generally include (i) a container holding a pharmaceutical composition that comprises at least one VRl modulator as described herein and (ii) instructions (e.g., labeling or a package insert) indicating that the contained composition is to be used for treating a condition responsive to VRl modulation in the patient.
  • VRl modulators provided herein may be used to alter activity and/or activation of capsaicin receptors in a variety of contexts, both in vitro and in vivo.
  • VRl antagonists may be used to inhibit the binding of vanilloid ligand agonist (such as capsaicin and/or RTX) to capsaicin receptor in vitro or in vivo.
  • such methods comprise the step of contacting a capsaicin receptor with one or more VRl modulators provided herein, in the presence of vanilloid ligand in aqueous solution and under conditions otherwise suitable for binding of the ligand to capsaicin receptor.
  • the VRl modulator(s) are generally present at a concentration that is sufficient to alter the binding of vanilloid ligand to VRl in vitro (using the assay provided in Example 5) and/or VRl -mediated signal transduction (using an assay provided in Example 6).
  • the capsaicin receptor may be present in solution or suspension ⁇ e.g., in an isolated membrane or cell preparation), or in a cultured or isolated cell. Within certain embodiments, the capsaicin receptor is expressed by a neuronal cell present in a patient, and the aqueous solution is a body fluid.
  • one or more VRl modulators are administered to an animal in an amount such that the VRl modulator is present in at least one body fluid of the animal at a therapeutically effective concentration that is 1 micromolar or less; preferably 500 nanomolar or less; more preferably 100 nanomolar or less, 50 nanomolar or less, 20 nanomolar or less, or 10 nanomolar or less.
  • a therapeutically effective dose that is less than 20 mg/kg body weight, preferably less than 5 mg/kg and, in some instances, less than 1 ⁇ g/kg.
  • modulation may be achieved by contacting a capsaicin receptor (either in vitro or in vivo) with one or more VRl modulators provided herein under conditions suitable for binding of the modulator(s) to the receptor.
  • the VRl modulator(s) are generally present at a concentration that is sufficient to alter the binding of vanilloid ligand to VRl in vitro and/or VRl -mediated signal transduction as described herein.
  • the receptor may be present in solution or suspension, in a cultured or isolated cell preparation or in a cell within a patient.
  • the cell may be a neuronal cell that is contacted in vivo in an animal.
  • the cell may be an epithelial cell, such as a urinary bladder epithelial cell (urothelial cell) or an airway epithelial cell that is contacted in vivo in an animal.
  • Modulation of signal tranducing activity may be assessed by detecting an effect on calcium ion conductance (also referred to as calcium mobilization or flux).
  • Modulation of signal transducing activity may alternatively be assessed by detecting an alteration of a symptom ⁇ e.g., pain, burning sensation, broncho-constriction, inflammation, cough, hiccup, itch, urinary incontinence or overactive bladder) of a patient being treated with one or more VRl modulators provided herein.
  • a symptom e.g., pain, burning sensation, broncho-constriction, inflammation, cough, hiccup, itch, urinary incontinence or overactive bladder
  • VRl modulator(s) provided herein are preferably administered to a patient ⁇ e.g., a human) orally or topically, and are present within at least one body fluid of the animal while modulating VRl signal-transducing activity.
  • Preferred VRl modulators for use in such methods modulate VRl signal-transducing activity in vitro at a concentration of 1 nanomolar or less, preferably 100 picomolar or less, more preferably 20 picomolar or less, and in vivo at a concentration of 1 micromolar or less, 500 nanomolar or less, or 100 nanomolar or less in a body fluid such as blood.
  • the present invention further provides methods for treating conditions responsive to VRl modulation.
  • treatment encompasses both disease-modifying treatment and symptomatic treatment, either of which may be prophylactic (i.e., before the onset of symptoms, in order to prevent, delay or reduce the severity of symptoms) or therapeutic (i.e., after the onset of symptoms, in order to reduce the severity and/or duration of symptoms).
  • a condition is "responsive to VRl modulation” if it is characterized by inappropriate activity of a capsaicin receptor, regardless of the amount of vanilloid ligand present locally, and/or if modulation of capsaicin receptor activity results in alleviation of the condition or a symptom thereof.
  • Such conditions include, for example, symptoms resulting from exposure to VRl- activating stimuli, pain, respiratory disorders (such as cough, asthma, chronic obstructive pulmonary disease, chronic bronchitis, cystic fibrosis and rhinitis, including allergic rhinitis, such as seasonal an perennial rhinitis, and non-allergic rhinitis), depression, itch, urinary incontinence, overactive bladder, hiccup and obesity, as described in more detail below.
  • Such conditions may be diagnosed and monitored using criteria that have been established in the art. Patients may include humans, domesticated companion animals and livestock, with dosages as described above.
  • Treatment regimens may vary depending on the compound used and the particular condition to be treated; however, for treatment of most disorders, a frequency of administration of 4 times daily or less is preferred. In general, a dosage regimen of 2 times daily is more preferred, with once a day dosing particularly preferred. For the treatment of acute pain, a single dose that rapidly reaches effective concentrations is desirable. It will be understood, however, that the specific dose level and treatment regimen for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy. In general, the use of the minimum dose sufficient to provide effective therapy is preferred. Patients may generally be monitored for therapeutic effectiveness using medical or veterinary criteria suitable for the condition being treated or prevented.
  • Patients experiencing symptoms resulting from exposure to capsaicin receptor-activating stimuli include individuals with burns caused by heat, light, tear gas or acid and those whose mucous membranes are exposed (e.g., via ingestion, inhalation or eye contact) to capsaicin (e.g., from hot peppers or in pepper spray) or a related irritant such as acid, tear gas, infectious agent(s) or air pollutant(s).
  • the resulting symptoms may include, for example, pain, broncho-constriction and inflammation.
  • Pain that may be treated using the VRl modulators provided herein may be chronic or acute and includes, but is not limited to, peripheral nerve-mediated pain (especially neuropathic pain).
  • Compounds provided herein may be used in the treatment of, for example, postmastectomy pain syndrome, stump pain, phantom limb pain, oral neuropathic pain, toothache (dental pain), denture pain, postherpetic neuralgia, diabetic neuropathy, chemotherapy-induced neuropathy, reflex sympathetic dystrophy, trigeminal neuralgia, osteoarthritis, rheumatoid arthritis, fibromyalgia, Guillain-Barre syndrome, meralgia paresthetica, burning-mouth syndrome and/or pain associated with nerve and root damage, including as pain associated with peripheral nerve disorders ⁇ e.g., nerve entrapment and brachial plexus avulsions, amputation, peripheral neuropathies including bilateral peripheral neuropathy, tic douloureux, atypical facial pain, nerve root damage, and
  • Additional neuropathic pain conditions include causalgia (reflex sympathetic dystrophy - RSD, secondary to injury of a peripheral nerve), neuritis (including, for example, sciatic neuritis, peripheral neuritis, polyneuritis, optic neuritis, postfebrile neuritis, migrating neuritis, segmental neuritis and Gombault's neuritis), neuronitis, neuralgias ⁇ e.g., those mentioned above, cervicobrachial neuralgia, cranial neuralgia, geniculate neuralgia, glossopharyngial neuralgia, migranous neuralgia, idiopathic neuralgia, intercostals neuralgia, mammary neuralgia, mandibular joint neuralgia, Morton's neuralgia, nasociliary neuralgia, occipital neuralgia, red neuralgia, Sluder's neuralgia, splenopalatine neural
  • Headache including headaches involving peripheral nerve activity may also be treated as described herein.
  • pain includes, for example, such as sinus, cluster ⁇ i.e., migranous neuralgia) and tension headaches, migraine, temporomandibular pain and maxillary sinus pain.
  • migraine headaches may be prevented by administration of a compound provided herein as soon as a pre-migrainous aura is experienced by the patient.
  • Additional pain conditions that may be treated as described herein include pain associated with respiratory disorders as described above, autoimmune diseases, immunodeficiency disorders, hot flashes, inflammatory bowel disease, gastroesophageal reflux disease (GERD), irritable bowel syndrome and/or inflammatory bowel disease.
  • VRl modulators provided herein may be used for the treatment of mechanical pain.
  • mechanical pain refers to pain other than headache pain that is not neuropathic or a result of exposure to heat, cold or external chemical stimuli.
  • Mechanical pain includes physical trauma (other than thermal or chemical burns or other irritating and/or painful exposures to noxious chemicals) such as post-surgical pain and pain from cuts, bruises and broken bones; toothache; denture pain; nerve root pain; osteoarthritis; rheumatoid arthritis; fibromyalgia; meralgia paresthetica; back pain; cancer-associated pain; angina; carpel tunnel syndrome; and pain resulting from bone fracture, labor, hemorrhoids, intestinal gas, dyspepsia, and menstruation.
  • Urinary tract conditions that may be treated as described herein include urinary incontinence (including overflow incontinence, urge incontinence and stress incontinence), as well as overactive or unstable bladder conditions (including bladder detrusor hyper-reflexia, detrusor hyper-reflexia of spinal origin and bladder hypersensitivity), m certain such treatment methods,
  • VRl modulator is administered via a catheter or similar device, resulting in direct injection of VRl modulator into the bladder.
  • Compounds provided herein may also be used as anti-tussive agents
  • VRl modulators provided herein may be used within combination therapy for the treatment of conditions involving pain and/or inflammatory components.
  • Such conditions include, for example, autoimmune disorders and pathologic autoimmune responses known to have an inflammatory component including, but not limited to, arthritis (especially rheumatoid arthritis), psoriasis, Crohn's disease, lupus erythematosus, irritable bowel syndrome, tissue graft rejection, and hyperacute rejection of transplanted organs.
  • Other such conditions include trauma (e.g., injury to the head or spinal cord), cardio- and cerebro-vascular disease and certain infectious diseases.
  • a VRl modulator is administered to a patient along with an analgesic and/or anti-inflammatory agent.
  • the VRl modulator and analgesic and/or antiinflammatory agent may be present in the same pharmaceutical composition, or may be administered separately in either order.
  • Anti-inflammatory agents include, for example, non- steroidal anti-inflammatory drugs (NSAIDs), non-specific and cyclooxygenase-2 (COX-2) specific cyclooxgenase enzyme inhibitors, gold compounds, corticosteroids, methotrexate, tumor necrosis factor (TNF) receptor antagonists, anti-TNF alpha antibodies, anti-C5 antibodies, and interleukin-1 (IL-I) receptor antagonists.
  • NSAIDs non- steroidal anti-inflammatory drugs
  • COX-2 non-specific and cyclooxygenase-2
  • IL-I interleukin-1
  • NSADDs include, but are not limited to ibuprofen (e.g., ADVILTM, MOTRINTM), flurbiprofen (ANSAIDTM), naproxen or naproxen sodium (e.g., NAPROSYN, ANAPROX, ALEVETM), diclofenac (e.g., CATAFLAMTM, VOLTARENTM), combinations of diclofenac sodium and misoprostol (e.g., ARTHROTECTM), sulindac (CUNOR ⁇ TM), oxaprozin (DAYPROTM), diflunisal (DOLOBIDTM), piroxicam (FELDENETM), indomethacin (ESTDOCINTM), etodolac (LODINETM), fenoprofen calcium (NALFONTM), ketoprofen (e.g., ORUDISTM, ORUVAILTM), sodium nabumetone (RELAFENTM), sulfasalazine (
  • NSAIDs consists of compounds that inhibit cyclooxygenase (COX) enzymes; such compounds include celecoxib (CELEBREXTM) and rofecoxib (VIOXXTM). NSAIDs further include salicylates such as acetylsalicylic acid or aspirin, sodium salicylate, choline and magnesium salicylates (TRILISATETM), and salsalate (DISALCIDTM), as well as corticosteroids such as cortisone (CORTONETM acetate), dexamethasone (e.g., DECADRONTM), methylprednisolone (MEDROLTM), prednisolone (PRELONETM), prednisolone sodium phosphate (PEDIAPREDTM), and prednisone (e.g., PREDNICEN-MTM, DELTASONETM, STERAPREDTM). Further anti-inflammatory agents include meloxicam, rofecoxib, celecoxib, e
  • Suitable dosages for VRl modulator within such combination therapy are generally as described above. Dosages and methods of administration of anti-inflammatory agents can be found, for example, in the manufacturer's instructions in the Physician's Desk Reference.
  • the combination administration of a VRl modulator with an anti- inflammatory agent results in a reduction of the dosage of the anti-inflammatory agent required to produce a therapeutic effect (i.e., a decrease in the minimum therapeutically effective amount).
  • the dosage of anti-inflammatory agent in a combination or combination treatment method is less than the maximum dose advised by the manufacturer for administration of the antiinflammatory agent without combination administration of a VRl antagonist.
  • this dosage is less than 3 A, even more preferably less than 1 A, and highly preferably, less than 1 A of the maximum dose, while most preferably the dose is less than 10% of the maximum dose advised by the manufacturer for administration of the anti-inflammatory agent(s) when administered without combination administration of a VRl antagonist. It will be apparent that the dosage amount of VRl antagonist component of the combination needed to achieve the desired effect may similarly be affected by the dosage amount and potency of the anti-inflammatory agent component of the combination.
  • the combination administration of a VRl modulator with an anti-inflammatory agent is accomplished by packaging one or more VRl modulators and one or more anti-inflammatory agents in the same package, either in separate containers within the package or in the same contained as a mixture of one or more VRl antagonists and one or more anti-inflammatory agents.
  • Preferred mixtures are formulated for oral administration (e.g., as pills, capsules, tablets or the like).
  • the package comprises a label bearing indicia indicating that the one or more VRl modulators and one or more anti-inflammatory agents are to be taken together for the treatment of an inflammatory pain condition.
  • VRl modulators provided herein may be used in combination with one or more additional pain relief medications.
  • Certain such medications are also anti- inflammatory agents, and are listed above.
  • Other such medications are analgesic agents, including narcotic agents which typically act at one or more opioid receptor subtypes (e.g., ⁇ , K and/or ⁇ ), preferably as agonists or partial agonists.
  • opioid receptor subtypes e.g., ⁇ , K and/or ⁇
  • Such agents include opiates, opiate derivatives and opioids, as well as pharmaceutically acceptable salts and hydrates thereof.
  • narcotic analgesics include, within preferred embodiments, alfentanil, alphaprodine, anileridine, bezitramide, buprenorphine, butorphanol, codeine, diacetyldihydromorphine, diacetylmorphine, dihydrocodeine, diphenoxylate, ethylmorphine, fentanyl, heroin, hydrocodone, hydromorphone, isomethadone, levomethorphan, levorphane, levorphanol, meperidine, metazocine, methadone, methorphan, metopon, morphine, nalbuphine, opium extracts, opium fluid extracts, powdered opium, granulated opium, raw opium, tincture of opium, oxycodone, oxymorphone, paregoric, pentazocine, pethidine, phenazocine, piminodine, propoxyphene, racemethorphan
  • narcotic analgesic agents include acetorphine, acetyldihydrocodeine, acetylmethadol, allylprodine, alphracetylmethadol, alphameprodine, alphamethadol, benzethidine, benzylmorphine, betacetylmethadol, betameprodine, betamethadol, betaprodine, clonitazene, codeine methylbromide, codeine-N-oxide, cyprenorphine, desomorphine, dextromoramide, diampromide, diethylthiambutene, dihydromorphine, dimenoxadol, dimepheptanol, dimethylthiamubutene, dioxaphetyl butyrate, dipipanone, drotebanol, ethanol, ethylmethylthiambutene, etonitazene, e
  • analgesic agents include, for example acetaminophen (paracetamol); aspirin and other NSAIDs described above; NR2B antagonists; bradykinin antagonists; anti-migraine agents; anticonvulsants such as oxcarbazepine and carbamazepine; antidepressants (such as TCAs, SSRIs, SNRIs, substance P antagonists, etc.); spinal blocks; gabapentin; asthma treatments (such as ⁇ 2 -adrenergic receptor agonists; leukotriene D 4 antagonists (e.g., montelukast); TALWIN® Nx and DEMEROL® (both available from Sanof ⁇ Winthrop Pharmaceuticals; New York, NY); LEVO-DROMORAN®; BUPRENEX® (Reckitt & Coleman Pharmaceuticals, Inc.; Richmond, VA); MSIR® (Purdue Pharma L.P.; Norwalk, CT); DILAUDID® (Knoll Pharmaceutical Co
  • Still further analgesic agents include CB2- receptor agonists, such as AM 1241, and compounds that bind to the o2 ⁇ subunit, such as Neurontin (Gabapentin) and pregabalin.
  • Representative anti-migraine agents for use in combination with a VRl modulator provided herein include CGRP antagonists, ergotamines and 5-HTi agonists, such as sumatripan, naratriptan, zolmatriptan and rizatriptan.
  • VRl modulators provided herein may be used in combination with one or more leukotriene receptor antagonists (e.g., agents that inhibits the cysteinyl leukotriene CySLT 1 receptor).
  • CysLTi antagonists include Montelukast (SESfGULAIR®; Merck & Co., Inc.). Such combinations find use in the treatment of pulmonary disorders such as asthma.
  • a VRl modulator as provided herein may be used in combination with other medication designed to treat this condition, such as antibiotics, anti-inflammatory agents, cystinyl leukotrienes, histamine antagonists, corticosteroids, opioids, NMDA antagonists, proton pump inhibitors, nociceptin, neurokinin (NKl, NK2 and NK3) and bradykinin (BKl and BK2) receptor antagonists, cannabinoids, blockers of Na+-dependent channels and large conductance Ca +2 -dependent K + -channel activators.
  • other medication designed to treat this condition such as antibiotics, anti-inflammatory agents, cystinyl leukotrienes, histamine antagonists, corticosteroids, opioids, NMDA antagonists, proton pump inhibitors, nociceptin, neurokinin (NKl, NK2 and NK3) and bradykinin (BKl and BK2) receptor antagonists, cannabinoids, blockers of Na+-dependent channels and large conductance Ca
  • Specific agents include dexbrompheniramine plus pseudoephedrine, loratadine, oxymetazoline, ipratropium, albuterol, beclomethasone, morphine, codeine, pholcodeine and dextromethorphan.
  • the present invention further provides combination therapy for the treatment of urinary incontinence.
  • a VRl modulator provided herein may be used in combination with other medication designed to treat this condition, such as estrogen replacement therapy, progesterone congeners, electrical stimulation, calcium channel blockers, antispasmodic agents, cholinergic antagonists, antimuscarinic drugs, tricyclic antidepressants, SNRIs, beta adrenoceptor agonists, phosphodiesterase inhibitors, potassium channel openers, nociceptin/orphanin FQ (OP4) agonists, neurokinin (NKl and NK2) antagonists, P2X3 antagonists, musculotrophic drugs and sacral neuromodulation.
  • other medication designed to treat this condition, such as estrogen replacement therapy, progesterone congeners, electrical stimulation, calcium channel blockers, antispasmodic agents, cholinergic antagonists, antimuscarinic drugs, tricyclic antidepressants, SNRIs, beta adrenoceptor agonists,
  • Specific agents include oxybutinin, emepronium, tolterodine, flavoxate, flurbiprofen, tolterodine, dicyclomine, propiverine, propantheline, dicyclomine, imipramine, doxepin, duloxetine, l-deamino-8-D-arginine vasopressin, muscarinic receptor antagonists such as Tolterodine (DETROL®; Pharmacia Corporation) and anticholinergic agents such as Oxybutynin (DITROPAN®; Ortho-McNeil Pharmaceutical, Inc., Raritan, NJ).
  • Suitable dosages for VRl modulator within such combination therapy are generally as described above.
  • compositions as described above may further comprise one or more additional medications as described above.
  • the additional medication is an analgesic.
  • packaged pharmaceutical preparations comprising one or more VRl modulators and one or more additional medications (e.g., analgesics) in the same package.
  • packaged pharmaceutical preparations generally include (i) a container holding a pharmaceutical composition that comprises at least one VRl modulator as described herein; (ii) a container holding a pharmaceutical composition that comprises at least one additional medication (such as a pain relief and/or anti-inflammatory medication) as described above and (iii) instructions (e.g., labeling or a package insert) indicating that the compositions are to be used simultaneously, separately or sequentially for treating or preventing a condition responsive to VRl modulation in the patient (such as a condition in which pain and/or inflammation predominates).
  • VRl agonists may further be used, for example, in crowd control (as a substitute for tear gas) or personal protection (e.g., in a spray formulation) or as pharmaceutical agents for the treatment of pain, itch, urinary incontinence or overactive bladder via capsaicin receptor desensitization.
  • crowd control as a substitute for tear gas
  • personal protection e.g., in a spray formulation
  • pharmaceutical agents for the treatment of pain, itch, urinary incontinence or overactive bladder via capsaicin receptor desensitization are formulated and used according to conventional tear gas or pepper spray technology.
  • the present invention provides a variety of non-pharmaceutical in vitro and in vivo uses for the compounds provided herein.
  • such compounds may be labeled and used as probes for the detection and localization of capsaicin receptor (in samples such as cell preparations or tissue sections, preparations or fractions thereof).
  • compounds provided herein that comprise a suitable reactive group such as an aryl carbonyl, nitro or azide group may be used in photoaffmity labeling studies of receptor binding sites.
  • compounds provided herein may be used as positive controls in assays for receptor activity, as standards for determining the ability of a candidate agent to bind to capsaicin receptor, or as radiotracers for positron emission tomography (PET) imaging or for single photon emission computerized tomography (SPECT).
  • PET positron emission tomography
  • SPECT single photon emission computerized tomography
  • Such methods can be used to characterize capsaicin receptors in living subjects.
  • a VRl modulator may be labeled using any of a variety of well known techniques (e.g., radiolabeled with a radionuclide such as tritium, as described herein), and incubated with a sample for a suitable incubation time (e.g., determined by first assaying a time course of binding).
  • unbound compound is removed (e.g., by washing), and bound compound detected using any method suitable for the label employed (e.g., autoradiography or scintillation counting for radiolabeled compounds; spectroscopic methods may be used to detect luminescent groups and fluorescent groups).
  • any method suitable for the label employed e.g., autoradiography or scintillation counting for radiolabeled compounds; spectroscopic methods may be used to detect luminescent groups and fluorescent groups.
  • a matched sample containing labeled compound and a greater (e.g., 10-fold greater) amount of unlabeled compound may be processed in the same manner. A greater amount of detectable label remaining in the test sample than in the control indicates the presence of capsaicin receptor in the sample.
  • Detection assays including receptor autoradiography (receptor mapping) of capsaicin receptor in cultured cells or tissue samples may be performed as described by Kuhar in sections 8.1.1 to 8.1.9 of Current Protocols in Pharmacology (1998) John Wiley & Sons, New York.
  • modulators may be linked to the interior surface of a tissue culture plate or other support, for use as affinity ligands for immobilizing and thereby isolating, capsaicin receptors ⁇ e.g., isolating receptor-expressing cells) in vitro.
  • a modulator linked to a fluorescent marker such as fluorescein, is contacted with the cells, which are then analyzed (or isolated) by fluorescence activated cell sorting (FACS).
  • VRl modulators provided herein may further be used within assays for the identification of other agents that bind to capsaicin receptor.
  • assays are standard competition binding assays, in which bound, labeled VRl modulator is displaced by a test compound.
  • such assays are performed by: (a) contacting capsaicin receptor with a radiolabeled VRl modulator as described herein, under conditions that permit binding of the VRl modulator to capsaicin receptor, thereby generating bound, labeled VRl modulator; (b) detecting a signal that corresponds to the amount of bound, labeled VRl modulator in the absence of test agent; (c) contacting the bound, labeled VRl modulator with a test agent; (d) detecting a signal that corresponds to the amount of bound labeled VRl modulator in the presence of test agent; and (e) detecting a decrease in signal detected in step (d), as compared to the signal detected in step (b).
  • Mass spectroscopy data in this and the following Examples is Electrospray MS, obtained in positive ion mode using a Micromass Time-of-Flight LCT (Micromass, Beverly MA), equipped with a Waters 600 pump (Waters Corp., Milford, MA), Waters 996 photodiode array detector, Gilson 215 autosampler (Gilson, Inc. Middleton, WI), and a Gilson 841 microinjector. MassLynx (Advanced Chemistry Development, Inc; Toronto, Canada) version 4.0 software with OpenLynx processing is used for data collection and analysis.
  • Micromass Time-of-Flight LCT Micromass, Beverly MA
  • Waters 600 pump Waters Corp., Milford, MA
  • Gilson 215 autosampler Gilson, Inc. Middleton, WI
  • Gilson 841 microinjector MassLynx (Advanced Chemistry Development, Inc; Toronto, Canada) version 4.0 software with OpenLynx processing is used for data collection and analysis.
  • This Example illustrates the preparation of representative heteroaryl substituted piperazinyl-pyridine analogues.
  • This Example illustrates the preparation of additional representative heteroaryl substituted piperazinyl-pyridine analogues.
  • This compound is prepared from 2,4-dichloropyrimidine and 4-bromo-2-chloro-l-fluoro- benzene using a procedure analogous to that used for the preparation of 2,4-dichloro-6-(4- fluorophenyl)pyrimidine in Example IA, step 2.
  • the IC 50 determined as described in Example 6 is less than 1 micromolar.
  • a cDNA encoding full length human capsaicin receptor (SEQ ID NO:1, 2 or 3 of U.S. Patent No. 6,482,611) is subcloned in the plasmid pBK-CMV (Stratagene, La MIa, CA) for recombinant expression in mammalian cells.
  • Human embryonic kidney (HEK293) cells are transfected with the pBK-CMV expression construct encoding the full length human capsaicin receptor using standard methods. The transfected cells are selected over two weeks in media containing G418 (400 ⁇ g/ml) to obtain a pool of stably transfected cells.
  • Previously frozen cells are disrupted with the aid of a tissue homogenizer in ice-cold HEPES homogenization buffer (5mM KCl 5, 5.8mM NaCl, 0.75mM CaCl 2 , 2mM MgCl 2 , 320 mM sucrose, and 10 mM HEPES pH 7.4).
  • Tissue homogenates are first centrifuged for 10 minutes at 1000 x g (4°C) to remove the nuclear fraction and debris, and then the supernatant from the first centrifugation is further centrifuged for 30 minutes at 35,000 x g (4°C) to obtain a partially purified membrane fraction.
  • Membranes are resuspended in the HEPES homogenization buffer prior to the assay.
  • This Example illustrates a representative assay of capsaicin receptor binding that may be used to determine the binding affinity of compounds for the capsaicin (VRl) receptor.
  • Binding studies with [ 3 H] Resiniferatoxin (RTX) are carried out essentially as described by Szallasi and Blumberg (1992) J. Pharmacol. Exp. Ter. 2(52: 883-888. In this protocol, nonspecific RTX binding is reduced by adding bovine alpha] acid glycoprotein (100 ⁇ g per tube) after the binding reaction has been terminated.
  • RTX (37 Ci/mmol) is synthesized by and obtained from the Chemical Synthesis and Analysis Laboratory, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD. [ 3 H] RTX may also be obtained from commercial vendors (e.g., Amersham Pharmacia Biotech, Inc.; Piscataway, NJ).
  • the membrane homogenate of Example 4 is centrifuged as before and resuspended to a protein concentration of 333 ⁇ g/ml in homogenization buffer. Binding assay mixtures are set up on ice and contain [ 3 H]RTX (specific activity 2200 mCi/ml), 2 ⁇ l non-radioactive test compound, 0.25 mg/ml bovine serum albumin (Cohn fraction V), and 5 x 10 4 - 1 x 10 5 VRl-transfected cells. The final volume is adjusted to 500 ⁇ l (for competition binding assays) or 1,000 ⁇ l (for saturation binding assays) with the ice-cold HEPES homogenization buffer solution (pH 7.4) described above.
  • [ 3 H]RTX specific activity 2200 mCi/ml
  • 2 ⁇ l non-radioactive test compound 0.25 mg/ml bovine serum albumin (Cohn fraction V)
  • 5 x 10 4 - 1 x 10 5 VRl-transfected cells The final volume is adjusted
  • Non-specific binding is defined as that occurring in the presence of 1 ⁇ M non-radioactive RTX (Alexis Corp.; San Diego, CA).
  • RTX non-radioactive RTX
  • [ 3 H]RTX is added in the concentration range of 7-1,000 pM, using 1 to 2 dilutions. Typically 11 concentration points are collected per saturation binding curve.
  • This Example illustrates representative calcium mobilization assays for use in evaluating test compounds for agonist and antagonist activity.
  • Cells transfected with expression plasmids (as described in Example 4) and thereby expressing human capsaicin receptor are seeded and grown to 70-90% confluency in FALCON black-walled, clear-bottomed 96-well plates (#3904, BECTON-DICKINSON, Franklin Lakes, NJ).
  • the culture medium is emptied from the 96 well plates and FLUO-3 AM calcium sensitive dye (Molecular Probes, Eugene, OR) is added to each well (dye solution: 1 mg FLUO-3 AM, 440 ⁇ L DMSO and 440 ⁇ l 20% pluronic acid in DMSO, diluted 1 :250 in Krebs-Ringer HEPES (KRH) buffer (25 mM HEPES, 5 mM KCl, 0.96 mM NaH 2 PO 4 , 1 mM MgSO 4 , 2 mM CaCl 2 , 5 mM glucose, 1 mM probenecid, pH 7.4), 50 ⁇ l diluted solution per well).
  • KRH Krebs-Ringer HEPES
  • Plates are covered with aluminum foil and incubated at 37 0 C for 1-2 hours in an environment containing 5% CO 2 . After the incubation, the dye is emptied from the plates, and the cells are washed once with KRH buffer, and resuspended in KRH buffer.
  • the EC 50 of the ago ⁇ iist capsaicin is first determined. An additional 20 ⁇ l of KRH buffer and 1 ⁇ l DMSO is added to each well of cells, prepared as described above. 100 ⁇ l capsaicin in KRH buffer is automatically transferred by the FLIPR instrument to each well. Capsaicin-induced calcium mobilization is monitored using either FLUOROSKAN ASCENT (Labsystems; Franklin, MA) or FLIPR (fluorometric imaging plate reader system; Molecular Devices, Sunnyvale, CA) instruments.
  • y is the maximum fluorescence signal
  • x is the concentration of the agonist or antagonist (in this case, capsaicin)
  • a is the E m3x
  • b corresponds to the EC 50 value
  • c is the Hill coefficient.
  • Test compounds are dissolved in DMSO, diluted in KRH buffer, and immediately added to cells prepared as described above. 100 nM capsaicin (an approximate EC 90 concentration) is also added to cells in the same 96-well plate as a positive control. The final concentration of test compounds in the assay wells is between 0.1 nM and 5 ⁇ M.
  • the ability of a test compound to act as an agonist of the capsaicin receptor is determined by measuring the fluorescence response of cells expressing capsaicin receptors elicited by the compound as function of compound concentration. This data is fit as described above to obtain the EC 50 , which is generally less than 1 micromolar, preferably less than 100 nM, and more preferably less than 10 nM.
  • the extent of efficacy of each test compound is also determined by calculating the response elicited by a concentration of test compound (typically 1 ⁇ M) relative to the response elicited by 100 nM capsaicin. This value, called Percent of Signal (POS), is calculated by the following equation:
  • This analysis provides quantitative assessment of both the potency and efficacy of test compounds as human capsaicin receptor agonists.
  • Agonists of the human capsaicin receptor generally elicit detectable responses at concentrations less than 100 ⁇ M, or preferably at concentrations less than 1 ⁇ M, or most preferably at concentrations less than 10 nM.
  • Extent of efficacy at human capsaicin receptor is preferably greater than 30 POS, more preferably greater than 80 POS at a concentration of 1 ⁇ M.
  • Certain agonists are essentially free of antagonist activity as demonstrated by the absence of detectable antagonist activity in the assay described below at compound concentrations below 4 nM, more preferably at concentrations below 10 ⁇ M and most preferably at concentrations less than or equal to 100 ⁇ M.
  • Test compounds are dissolved in DMSO, diluted in 20 ⁇ l KRH buffer so that the final concentration of test compounds in the assay well is between 1 ⁇ M and 5 ⁇ M, and added to cells prepared as described above.
  • the 96 well plates containing prepared cells and test compounds are incubated in the dark, at room temperature for 0.5 to 6 hours. It is important that the incubation not continue beyond 6 hours.
  • 100 ⁇ l capsaicin in KRH buffer at twice the EC 50 concentration determined as described above is automatically added by the FLIPR instrument to each well of the 96 well plate for a final sample volume of 200 ⁇ l and a final capsaicin concentration equal to the EC 50 .
  • the final concentration of test compounds in the assay wells is between 1 ⁇ M and 5 ⁇ M.
  • Antagonists of the capsaicin receptor decrease this response by at least about 20%, preferably by at least about 50%, and most preferably by at least 80%, as compared to matched control (i.e., cells treated with capsaicin at twice the EC 50 concentration in the absence of test compound), at a concentration of 10 micromolar or less, preferably 1 micromolar or less.
  • the concentration of antagonist required to provide a 50% decrease, relative to the response observed in the presence of capsaicin and without antagonist, is the IC 50 for the antagonist, and is preferably below 1 micromolar, 100 nanomolar, 10 nanomolar or 1 nanomolar.
  • Certain preferred VRl modulators are antagonists that are essentially free of agonist activity as demonstrated by the absence of detectable agonist activity in the assay described above at compound concentrations below 4 nM, more preferably at concentrations below 10 ⁇ M and most preferably at concentrations less than or equal to 100 ⁇ M.
  • This Example illustrates the evaluation of compound half-life values (t 1/2 values) using a representative liver microsomal half-life assay.
  • liver microsomes are obtained from XenoTech LLC (Kansas City, KS). Such liver microsomes may also be obtained from In Vitro Technologies (Baltimore, MD) or Tissue Transformation Technologies (Edison, NJ). Six test reactions are prepared, each containing 25 ⁇ l microsomes, 5 ⁇ l of a 100 ⁇ M solution of test compound, and 399 ⁇ l 0.1 M phosphate buffer (19 niL 0.1 M NaH 2 PO 4 , 81 mL 0.1 M Na 2 HPO 4 , adjusted to pH 7.4 with H 3 PO 4 ).
  • a seventh reaction is prepared as a positive control containing 25 ⁇ l microsomes, 399 ⁇ l 0.1 M phosphate buffer, and 5 ⁇ l of a 100 ⁇ M solution of a compound with known metabolic properties (e.g., DIAZEPAM or CLOZAPINE). Reactions are preincubated at 39 0 C for 10 minutes.
  • a compound with known metabolic properties e.g., DIAZEPAM or CLOZAPINE
  • CoFactor Mixture is prepared by diluting 16.2 mg NADP and 45.4 mg Glucose-6- phosphate in 4 mL 100 mM MgCl 2 .
  • Glucose-6-phosphate dehydrogenase solution is prepared by diluting 214.3 ⁇ l glucose-6-phosphate dehydrogenase suspension (Roche Molecular Biochemicals; Indianapolis, IN) into 1285.7 ⁇ l distilled water.
  • 71 ⁇ l Starting Reaction Mixture (3 mL CoFactor Mixture; 1.2 mL Glucose-6-phosphate dehydrogenase solution) is added to 5 of the 6 test reactions and to the positive control.
  • 71 ⁇ l 100 mM MgCl 2 is added to the sixth test reaction, which is used as a negative control.
  • 75 ⁇ l of each reaction mix is pipetted into a well of a 96-well deep-well plate containing 75 ⁇ l ice-cold acetonitrile.
  • Samples are vortexed and centrifuged 10 minutes at 3500 rpm (Sorval T 6000D centrifuge, HlOOOB rotor).
  • 75 ⁇ l of supernatant from each reaction is transferred to a well of a 96-well plate containing 150 ⁇ l of a 0.5 ⁇ M solution of a compound with a known LCMS profile (internal standard) per well.
  • LCMS analysis of each sample is carried out and the amount of unmetabolized test compound is measured as AUC, compound concentration vs. time is plotted, and the ty 2 value of the test compound is extrapolated.
  • Preferred compounds provided herein exhibit in vitro t ]/2 values of greater than 10 minutes and less than 4 hours, preferably between 30 minutes and 1 hour, in human liver microsomes.
  • EXAMPLE 8 MDCK Toxicity Assay
  • This Example illustrates the evaluation of compound toxicity using a Madin Darby canine kidney (MDCK) cell cytotoxicity assay. 1 ⁇ L of test compound is added to each well of a clear bottom 96-well plate (PACKARD,
  • MDCK cells ATCC no. CCL-34 (American Type Culture Collection, Manassas, VA), are maintained in sterile conditions following the instructions in the ATCC production information sheet.
  • Confluent MDCK cells are trypsinized, harvested, and diluted to a concentration of 0.1 x 10 6 cells/ml with warm (37°C) medium (VITACELL Minimum Essential Medium Eagle, ATCC catalog # 30-2003). 100 ⁇ L of diluted cells is added to each well, except for five standard curve control wells that contain 100 ⁇ L of warm medium without cells. The plate is then incubated at 37°C under 95% O 2 , 5% CO 2 for 2 hours with constant shaking.
  • mammalian cell lysis solution from the PACKARD (Meriden, CT) ATP-LITE-M Luminescent ATP detection kit
  • PACKARD TOPSEAL stickers from the PACKARD (Meriden, CT) ATP-LITE-M Luminescent ATP detection kit
  • the ATP-LITE-M Luminescent ATP detection kit is generally used according to the manufacturer's instructions to measure ATP production in treated and untreated MDCK cells. PACKARD ATP LITE-M reagents are allowed to equilibrate to room temperature. Once equilibrated, the lyophilized substrate solution is reconstituted in 5.5 mL of substrate buffer solution (from kit). Lyophilized ATP standard solution is reconstituted in deionized water to give a 10 mM stock.
  • PACKARD substrate solution 50 ⁇ L is added to all wells, which are then covered, and the plates are shaken at approximately 700 rpm on a suitable shaker for 2 minutes.
  • a white PACKARD sticker is attached to the bottom of each plate and samples are dark adapted by wrapping plates in foil and placing in the dark for 10 minutes.
  • Luminescence is then measured at 22°C using a luminescence counter (e.g., PACKARD TOPCOUNT Microplate Scintillation and Luminescence Counter or TECAN SPECTRAFLUOR PLUS), and ATP levels calculated from the standard curve.
  • ATP levels in cells treated with test compound(s) are compared to the levels determined for untreated cells.
  • Cells treated with 10 ⁇ M of a preferred test compound exhibit ATP levels that are at least 80%, preferably at least 90%, of the untreated cells.
  • a 100 ⁇ M concentration of the test compound is used, cells treated with preferred test compounds exhibit ATP levels that are at least 50%, preferably at least 80%, of the ATP levels detected in untreated cells.
  • EXAMPLE 9 Dorsal Root Ganglion Cell Assay This Example illustrates a representative dorsal root ganglian cell assay for evaluating
  • VRl antagonist or agonist activity of a compound VRl antagonist or agonist activity of a compound.
  • DRG are dissected from neonatal rats, dissociated and cultured using standard methods
  • VRl -dependent increase in intracellular calcium levels which is monitored by a change in Fluo-4 fluorescence with a fluorometer. Data are collected for 60-180 seconds to determine the maximum fluorescent signal.
  • Antagonists of the capsaicin receptor preferably have an IC 50 below 1 micromolar, 100 nanomolar,
  • EC 50 or concentration required to achieve 50% of the maximum signal for a capsaicin-activated response, is preferably below 1 micromolar, below 100 nanomolar or below 10 nanomolar.
  • This Example illustrates representative methods for assessing the degree of pain relief provided by a compound.
  • the following methods may be used to assess pain relief.
  • Testing is initiated with the middle hair of the series with subsequent filaments being applied in consecutive fashion, ascending or descending, depending on whether a negative or positive response, respectively, is obtained with the initial filament.
  • Compounds are effective in reversing or preventing mechanical allodynia-like symptoms if rats treated with such compounds require stimulation with a Von Frey filament of higher rigidity strength to provoke a positive allodynic response as compared to control untreated or vehicle treated rats.
  • testing of an animal in chronic pain may be done before and after compound administration.
  • an effective compound results in an increase in the rigidity of the filament needed to induce a response after treatment, as compared to the filament that induces a response before treatment or in an animal that is also in chronic pain but is left untreated or is treated with vehicle.
  • Test compounds are administered before or after onset of pain. When a test compound is administered after pain onset, testing is performed 10 minutes to three hours after administration.
  • Test compound produces a reduction in mechanical hyperalgesia if there is a statistically significant decrease in the duration of hindpaw withdrawal.
  • Test compound may be administered before or after onset of pain. For compounds administered after pain onset, testing is performed 10 minutes to three hours after administration. THERMAL HYPERALGESIA
  • Thermal hyperalgesia an exaggerated response to noxious thermal stimulus
  • Hargreaves et al. (1988) Pain. 32(l):77-88 a constant radiant heat source is applied the animals' plantar surface of either hind paw.
  • the time to withdrawal i.e., the amount of time that heat is applied before the animal moves its paw
  • thermal threshold or latency determines the animal's hind paw sensitivity to heat.
  • Compounds produce a reduction in thermal hyperalgesia if there is a statistically significant increase in the time to hindpaw withdrawal (i.e., the thermal threshold to response or latency is increased).
  • Test compound may be administered before or after onset of pain. For compounds administered after pain onset, testing is performed 10 minutes to three hours after administration.
  • Pain may be induced using any of the following methods, to allow testing of analgesic efficacy of a compound.
  • compounds provided herein result in a statistically significant reduction in pain as determined by at least one of the previously described testing methods, using male SD rats and at least one of the following models.
  • Acute inflammatory pain is induced using the carrageenan model essentially as described by Field et al. (1997) Br. J. Pharmacol. 121(8):1513-1522. 100-200 ⁇ l of 1-2% carrageenan solution is injected into the rats' hind paw. Three to four hours following injection, the animals' sensitivity to thermal and mechanical stimuli is tested using the methods described above.
  • a test compound (0.01 to 50 mg/kg) is administered to the animal, prior to testing, or prior to injection of carrageenan. The compound can be administered orally or through any parenteral route, or topically on the paw. Compounds that relieve pain in this model result in a statistically significant reduction in mechanical allodynia and/or thermal hyperalgesia.
  • Chronic inflammatory pain is induced using one of the following protocols:
  • rats are tested for thermal hyperalgesia, mechanical allodynia and mechanical hyperalgesia as described above.
  • rats are tested on days 5, 6, and 7 following CFA injection.
  • animals are treated with a test compound, morphine or vehicle.
  • An oral dose of morphine of 1-5 mg/kg is suitable as positive control.
  • a dose of 0.01-50 mg/kg of test compound is used.
  • Compounds can be administered as a single bolus prior to testing or once or twice or three times daily, for several days prior to testing. Drugs are administered orally or through any parenteral route, or applied topically to the animal.
  • MPE Percent Maximum Potential Efficacy
  • Chronic neuropathic pain is induced using the chronic constriction injury (CCI) to the rat's sciatic nerve essentially as described by Bennett and Xie (1988) Pain 33:87-107.
  • Rats are anesthetized (e.g. with an intraperitoneal dose of 50-65 mg/kg pentobarbital with additional doses administered as needed).
  • the lateral aspect of each hind limb is shaved and disinfected.
  • an incision is made on the lateral aspect of the hind limb at the mid thigh level.
  • the biceps femoris is bluntly dissected and the sciatic nerve is exposed.
  • On the other side the sciatic nerve is not ligated and is not manipulated.
  • the muscle is closed with continuous pattern and the skin is closed with wound clips or sutures. Rats are assessed for mechanical allodynia, mechanical hyperalgesia and thermal hyperalgesia as described above.

Abstract

Substituted biaryl piperazinyl-pyridine analogues are provided, of the Formula: wherein variables are as described herein. Such compounds are ligands that may be used to modulate specific receptor activity in vivo or in vitro, and are particularly useful in the treatment of conditions associated with pathological receptor activation in humans, domesticated companion animals and livestock animals. Pharmaceutical compositions and methods for using such compounds to treat such disorders are provided, as are methods for using such ligands for receptor localization studies.

Description

HETEROARYL SUBSTITUTED PIPERAZINYL-PYRIDINE ANALOGUES
FIELD OF THE INVENTION
This invention relates generally to heteroaryl substituted piperazinyl-pyridine analogues that have useful pharmacological properties. The invention further relates to the use of such compounds for treating conditions related to capsaicin receptor activation, for identifying other agents that bind to capsaicin receptor, and as probes for the detection and localization of capsaicin receptors.
BACKGROUND OF THE INVENTION Pain perception, or nociception, is mediated by the peripheral terminals of a group of specialized sensory neurons, termed "nociceptors." A wide variety of physical and chemical stimuli induce activation of such neurons in mammals, leading to recognition of a potentially harmful stimulus. Inappropriate or excessive activation of nociceptors, however, can result in debilitating acute or chronic pain. Neuropathic pain involves pain signal transmission in the absence of stimulus, and typically results from damage to the nervous system. In most instances, such pain is thought to occur because of sensitization in the peripheral and central nervous systems following initial damage to the peripheral system (e.g., via direct injury or systemic disease). Neuropathic pain is typically burning, shooting and unrelenting in its intensity and can sometimes be more debilitating that the initial injury or disease process that induced it.
Existing treatments for neuropathic pain are largely ineffective. Opiates, such as morphine, are potent analgesics, but their usefulness is limited because of adverse side effects, such as physical addictiveness and withdrawal properties, as well as respiratory depression, mood changes, and decreased intestinal motility with concomitant constipation, nausea, vomiting, and alterations in the endocrine and autonomic nervous systems. In addition, neuropathic pain is frequently non-responsive or only partially responsive to conventional opioid analgesic regimens. Treatments employing the N-methyl-D-aspartate antagonist ketamine or the alpha(2)-adrenergic agonist clonidine can reduce acute or chronic pain, and permit a reduction in opioid consumption, but these agents are often poorly tolerated due to side effects. Topical treatment with capsaicin has been used to treat chronic and acute pain, including neuropathic pain. Capsaicin is a pungent substance derived from the plants of the Solanaceae family (which includes hot chili peppers) and appears to act selectively on the small diameter afferent nerve fibers (A-delta and C fibers) that are believed to mediate pain. The response to capsaicin is characterized by persistent activation of nociceptors in peripheral tissues, followed by eventual desensitization of peripheral nociceptors to one or more stimuli. From studies in animals, capsaicin appears to trigger C fiber membrane depolarization by opening cation selective channels for calcium and sodium.
Similar responses are also evoked by structural analogues of capsaicin that share a common vanilloid moiety. One such analogue is resiniferatoxin (RTX), a natural product of Euphorbia plants. The term vanilloid receptor (VR) was coined to describe the neuronal membrane recognition site for capsaicin and such related irritant compounds. The capsaicin response is competitively inhibited (and thereby antagonized) by another capsaicin analog, capsazepine, and is also inhibited by the non-selective cation channel blocker ruthenium red, which binds to VR with no more than moderate affinity (typically with a K; value of no lower than 140 μM). Rat and human vanilloid receptors have been cloned from dorsal root ganglion cells. The first type of vanilloid receptor to be identified is known as vanilloid receptor type 1 (VRl), and the terms "VRl" and "capsaicin receptor" are used interchangeably herein to refer to rat and/or human receptors of this type, as well as mammalian homologues. The role of VRl in pain sensation has been confirmed using mice lacking this receptor, which exhibit no vanilloid-evoked pain behavior and impaired responses to heat and inflammation. VRl is a nonselective cation channel with a threshold for opening that is lowered in response to elevated temperatures, low pH, and capsaicin receptor agonists. Opening of the capsaicin receptor channel is generally followed by the release of inflammatory peptides from neurons expressing the receptor and other nearby neurons, increasing the pain response. After initial activation by capsaicin, the capsaicin receptor undergoes a rapid desensitization via phosphorylation by cAMP-dependent protein kinase.
Because of their ability to desensitize nociceptors in peripheral tissues, VRl agonist vanilloid compounds have been used as topical anesthetics. However, agonist application may itself cause burning pain, which limits this therapeutic use. Recently, it has been reported that VRl antagonists, including certain nonvanilloid compounds, are also useful for the treatment of pain (see, e.g., PCT International Application Publication Numbers WO 02/08221, WO 03/062209, WO 04/054582, WO 04/055003, WO 04/055004, WO 04/056774, WO 05/007646, WO 05/007648, WO 05/007652, WO 05/009977, WO 05/009980 and WO 05/009982).
Thus, compounds that interact with VRl, but do not elicit the initial painful sensation of VRl agonist vanilloid compounds, are desirable for the treatment of chronic and acute pain, including neuropathic pain, as well as other conditions that are responsive to capsaicin receptor modulation. The present invention fulfills this need, and provides further related advantages. SUMMARY OF THE INVENTION
The present invention provides heteroaryl substituted piperazinyl-pyridine analogues of Formula I:
Figure imgf000004_0001
and pharmaceutically acceptable salts of such compounds. Within Formula I: Aτi is a 5-membered aromatic heterocycle that is substituted with from O to 4 substituents independently chosen from Ri; Ar2 is phenyl or a 6-membered aromatic heterocycle, each of which is optionally substituted, and is preferably substituted with from O to 4 substituents independently chosen from R2; W is CH or N; X, Y and Z are independently CRx or N, such that at least one of X, Y and Z is N;
Rx is independently chosen at each occurrence from hydrogen, Ci-Qalkyl, amino, cyano and mono- and di-(Ci-C4alkyl)amino; Each Ri is independently chosen from:
(a) halogen, cyano and nitro; (b) groups of the formula -Q-M-Ry; and
(c) groups that are taken together with an adjacent Ri to form a fused 5- to 7-membered carbocyclic or heterocyclic ring that is substituted with from O to 4 substituents independently chosen from halogen, cyano, nitro and groups of the formula -Q-M-Ry; Each Q is independently chosen from C0-C4alkylene (i.e., Q is absent or is a single covalent bond or a C]-C4alkylene group);
M is independently selected at each occurrence from a single covalent bond, O, C(=O), 0C(=0),
Ceθ)O, O-C(=O)O, S(0)m, N(R2), C(=O)N(RZ), C(=NH)N(RZ), N(RZ)C(=O), N(R2)C(=NH),
N(R2)S(O)n,, S(O)mN(Rz) and N[S(O)mR2]S(O)m; wherein m is independently selected at each occurrence from O, 1 and 2; and R2 is independently selected at each occurrence from hydrogen, C]-C8alkyl and groups that are taken together with Ry to form an optionally substituted 4- to 7-membered heterocycle; and
Each Ry is independently hydrogen, C]-C8haloalkyl, optionally substituted Ci-C8alkyl, optionally substituted (C3-C8carbocycle)C0-C4alkyl, optionally substituted (4- to 7-membered heterocycle)Co-C4alkyl, or taken together with R2 to form an optionally substituted 4- to 7- membered heterocycle, wherein each alkyl, carbocycle and heterocycle is preferably substituted with from 0 to 4 substituents independently selected from hydroxy, halogen, amino, cyano, nitro, oxo, -COOH, aminocarbonyl, CrC6alkyl, C3-C7cycloalkyl, C2-C6alkyl ether, CrC6alkanoyl, -SO2(CrC6alkyl), -SO2NH2, CrC8alkoxy, Ci-C8alkylthio, mono- and di- (CrC6alkyl)aminocarbonyl, mono- and di-(Ci-C6alkyl)amino and phenyl; such that Ry is not hydrogen if Q is Coalkyl and M is a single covalent bond; Each R2 is: (a) independently chosen from (i) hydroxy, amino, cyano, halogen, -COOH, -SO2NH2, nitro and aminocarbonyl; and (ii) Ci-C6alkyl, (C3-C8cycloalkyl)Co-C4alkyl, Ci-C6haloalkyl, Cr C6alkoxy, Ci-Cβalkylthio, C2-C6alkyl ether, C2-C6alkanoyl, CrC6alkoxycarbonyl, C2- C6alkanoyloxy, C3-C6alkanone, mono- and di-(Ci-C6alkyl)ammoC0-C6alkyl, mono- and di- (C3-Cscycloalkyl)aminoC0-C4alkyl, (4- to 7-membered heterocycle)C0-C4alkyl, Q- Qalkylsulfonyl, mono- and di-(CrC6alkyl)aminosulfonyl, and mono- and di-(Cr
C6alkyl)aminocarbonyl, each of which is optionally substituted, and is preferably substituted with from O to 4 substituents independently chosen from halogen, hydroxy, cyano, amino, -COOH and oxo; or
(b) taken together with an adjacent R2 to form a fused 5- to 13-membered carbocyclic or heterocyclic group that is optionally substituted, and is preferably substituted with from O to 3 substituents independently chosen from halogen, oxo and CrC6alkyl; R3 is selected from:
(i) hydrogen and halogen;
(ii) Ci-C6alkyl, (C3-C8cycloalkyl)Co-C2alkyl, CrC6haloalkyl and phenylC0-C2alkyl; and (iii) groups of the formula:
Λ^%β or Λ-%7 wherein:
L is Co-Cβalkylene or Ci-C6alkyl that is taken together with R5, R6 or R7 to form a 4- to 7- membered heterocycle; W is O, CO, S, SO or SO2;
R5 and R6 are:
(a) independently chosen from hydrogen, C]-Ci2alkyl, C2-Q2alkenyl, (C3- C8cycloalkyl)C0-C4alkyl, C2-C6alkanoyl, C,-C6alkylsulfonyl, phenylC0-C6alkyl, (4- to 7-membered heterocycle)C0-C6alkyl and groups that are joined to L to form a 4- to 7- membered heterocycle; or
(b) joined to form a 4- to 12-membered heterocycle; and
R7 is hydrogen, CrCi2alkyl, C2-C]2alkenyl, (C3-C8cycloalkyl)C0-C4alkyl, C2-C6alkanoyl, phenylCo-C6alkyl, (4- to 7-membered heterocycle)Co-C6alkyl or a group that is joined to L to form a 4- to 7-membered heterocycle; wherein each of (ii) and (iii) is optionally substituted, and is preferably substituted with from 0 to 4 substituents independently chosen from:
(1) halogen, hydroxy, amino, cyano, -COOH, -SO2NH2, oxo, nitro and aminocarbonyl; and (2) CrC6alkyl, (C3-C8cycloalkyl)Co-C2alkyl, C,-C6haloalkyl, CrC6alkoxy, C1-
Qalkoxycarbonyl, Q-Cghaloalkyl, Q-Cealkanoyl, C2-C6alkanoylamino, mono- and di-(Ci-C6alkyl)aminoCo-C4alkyl, Q-Cβalkylsulfonyl, mono- and di-(Cr
C6alkyl)aminosulfonyl, mono- and di-(Ci-Cealkyl)aminocarbonylCo-C4alkyl, phenylC0-C4alkyl and (4- to 7-membered heterocycle)C0-C4alkyl, each of which is substituted with from O to 4 secondary substituents independently chosen from halogen, hydroxy, cyano, oxo, imino, C]-C4alkyl, Ci-C4alkoxy and Ci-C4haloalkyl; and
R4 represents from 0 to 2 substituents that are preferably independently chosen from Ci-C3alkyl,
Ci-C3haloalkyl and oxo. Within certain aspects, compounds of Formula I are VRl modulators and exhibit a Kj of no greater than 1 micromolar, 500 nanomolar, 100 nanomolar, 50 nanomolar, 10 nanomolar or 1 nanomolar in a capsaicin receptor binding assay and/or have an EC50 or IC50 value of no greater than 1 micromolar, 500 nanomolar, 100 nanomolar, 50 nanomolar, 10 nanomolar or 1 nanomolar in an in vitro assay for determination of capsaicin receptor agonist or antagonist activity. In certain embodiments, such VRl modulators are VRl antagonists and exhibit no detectable agonist activity in an in vitro assay of capsaicin receptor activation (e.g., the assay provided in Example 6, herein) at a concentration equal to the IC50, 10 times the IC50 or 100 times the IC50.
Within certain aspects, compounds provided herein are labeled with a detectable marker (e.g., radiolabeled or fluorescein conjugated). The present invention further provides, within other aspects, pharmaceutical compositions comprising at least one compound of Formula I in combination with a physiologically acceptable carrier or excipient.
Within further aspects, methods are provided for reducing calcium conductance of a cellular capsaicin receptor, comprising contacting a cell (e.g., neuronal, such as cells of the central nervous system and/or peripheral ganglia, urothelial or lung) that expresses a capsaicin receptor with at least one VRl modulator as described herein. Such contact may occur in vivo or in vitro and is generally performed using a concentration of VRl modulator that is sufficient to alter the binding of vanilloid ligand to VRl in vitro (using the assay provided in Example 5) and/or VRl- mediated signal transduction (using an assay provided in Example 6). Methods are further provided for inhibiting binding of vanilloid ligand to a capsaicin receptor. Within certain such aspects, the inhibition takes place in vitro. Such methods comprise contacting a capsaicin receptor with at least one VRl modulator as described herein, under conditions and in an amount or concentration sufficient to detectably inhibit vanilloid ligand binding to the capsaicin receptor. Within other such aspects, the capsaicin receptor is in a patient. Such methods comprise contacting cells expressing a capsaicin receptor in a patient with at least one VRl modulator as described herein in an amount or concentration that would be sufficient to detectably inhibit vanilloid ligand binding to cells expressing a cloned capsaicin receptor in vitro.
The present invention further provides methods for treating a condition responsive to capsaicin receptor modulation in a patient, comprising administering to the patient a therapeutically effective amount of at least one VRl modulator as described herein.
Within other aspects, methods are provided for treating pain in a patient, comprising administering to a patient suffering from (or at risk for) pain a therapeutically effective amount of at least one VRl modulator as described herein.
Methods are further provided for treating itch, urinary incontinence, overactive bladder, cough and/or hiccup in a patient, comprising administering to a patient suffering from (or at risk for) one or more of the foregoing conditions a therapeutically effective amount of at least one VRl modulator as described herein.
The present invention further provides methods for promoting weight loss in an obese patient, comprising administering to an obese patient a therapeutically effective amount of at least one VRl modulator as described herein.
Methods are further provided for identifying an agent that binds to capsaicin receptor, comprising: (a) contacting capsaicin receptor with a labeled compound as described herein under conditions that permit binding of the compound to capsaicin receptor, thereby generating bound, labeled compound; (b) detecting a signal that corresponds to the amount of bound, labeled compound in the absence of test agent; (c) contacting the bound, labeled compound with a test agent; (d) detecting a signal that corresponds to the amount of bound labeled compound in the presence of test agent; and (e) detecting a decrease in signal detected in step (d), as compared to the signal detected in step (b).
Within further aspects, the present invention provides methods for determining the presence or absence of capsaicin receptor in a sample, comprising: (a) contacting a sample with a compound as described herein under conditions that permit binding of the compound to capsaicin receptor; and (b) detecting a signal indicative of a level of the compound bound to capsaicin receptor.
The present invention also provides packaged pharmaceutical preparations, comprising: (a) a pharmaceutical composition as described herein in a container; and (b) instructions for using the composition to treat one or more conditions responsive to capsaicin receptor modulation, such as pain, itch, urinary incontinence, overactive bladder, cough, hiccup and/or obesity.
In yet another aspect, the present invention provides methods for preparing the compounds disclosed herein, including the intermediates. These and other aspects of the invention will become apparent upon reference to the following detailed description.
DETAILED DESCRIPTION
As noted above, the present invention provides heteroaryl substituted piperazinyl-pyridine analogues. Such compounds may be used in vitro or in vivo, to modulate capsaicin receptor activity in a variety of contexts.
TERMINOLOGY
Compounds are generally described herein using standard nomenclature. For compounds having asymmetric centers, it should be understood that (unless otherwise specified) all of the optical isomers and mixtures thereof are encompassed. In addition, compounds with carbon- carbon double bonds may occur in Z- and E- forms, with all isomeric forms of the compounds being included in the present invention unless otherwise specified. Where a compound exists in various tautomeric forms, a recited compound is not limited to any one specific tautomer, but rather is intended to encompass all tautomeric forms. Certain compounds are described herein using a general formula that includes variables {e.g., Ri, X, A^). Unless otherwise specified, each variable within such a formula is defined independently of any other variable, and any variable that occurs more than one time in a formula is defined independently at each occurrence.
The term "heteroaryl substituted piperazinyl-pyridine analogue," as used herein, encompasses all compounds of Formula I, as well as compounds of other Formulas provided herein (including any enantiomers, racemates and stereoisomers) and pharmaceutically acceptable salts of such compounds. In other words, compounds in which the core ring
x R3 is pyridyl, pyrimidyl or triazinyl {e.g.,
Figure imgf000008_0001
Figure imgf000008_0002
Rs) are specifically included within the definition of substituted biaryl piperazinyl-pyridine analogues. A "pharmaceutically acceptable salt" of a compound is an acid or base salt that is generally considered in the art to be suitable for use in contact with the tissues of human beings or animals without excessive toxicity or carcinogenicity, and preferably without irritation, allergic response, or other problem or complication. Such salts include mineral and organic acid salts of basic residues such as amines, as well as alkali or organic salts of acidic residues such as carboxylic acids. Specific pharmaceutical salts include, but are not limited to, salts of acids such as hydrochloric, phosphoric, hydrobromic, malic, glycolic, fumaric, sulfuric, sulfamic, sulfanilic, formic, toluenesulfonic, methanesulfonic, benzene sulfonic, ethane disulfonic, 2- hydroxyethylsulfonic, nitric, benzoic, 2-acetoxybenzoic, citric, tartaric, lactic, stearic, salicylic, glutamic, ascorbic, pamoic, succinic, fumaric, maleic, propionic, hydroxymaleic, hydroiodic, phenylacetic, alkanoic such as acetic, HOOC-(CH2)n-COOH where n is 0-4, and the like. Similarly, pharmaceutically acceptable cations include, but are not limited to sodium, potassium, calcium, aluminum, lithium and ammonium. Those of ordinary skill in the art will recognize further pharmaceutically acceptable salts for the compounds provided herein, including those listed within Remington: The Science and Practice of Pharmacy, 21st ed., Lippincott Williams & Wilkins, Philadelphia, PA (2005). In general, a pharmaceutically acceptable acid or base salt can be synthesized from a parent compound that contains a basic or acidic moiety by any conventional chemical method. Briefly, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, the use of nonaqueous media, such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile, is preferred.
It will be apparent that each compound of Formula I may, but need not, be formulated as a hydrate, solvate or non-covalent complex. Ih addition, the various crystal forms and polymorphs are within the scope of the present invention. Also provided herein are prodrugs of the compounds of Formula I. A "prodrug" is a compound that may not fully satisfy the structural requirements of the compounds provided herein, but is modified in vivo, following administration to a patient, to produce a compound of Formula I, or other formula provided herein. For example, a prodrug may be an acylated derivative of a compound as provided herein. Prodrugs include compounds wherein hydroxy, amine or sulfhydryl groups are bonded to any group that, when administered to a mammalian subject, cleaves to form a free hydroxy, amino or sulfhydryl group, respectively. Examples of prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of alcohol and amine functional groups within the compounds provided herein. Prodrugs of the compounds provided herein may be prepared by modifying functional groups present in the compounds in such a way that the modifications are cleaved in vivo to yield the parent compounds.
As used herein, the term "alkyl" refers to a straight or branched chain saturated aliphatic hydrocarbon. Alkyl groups include groups having from 1 to 8 carbon atoms (CrC8alkyl), from 1 to 6 carbon atoms (Q-Cδalkyl) and from 1 to 4 carbon atoms (Ci-C4alkyl), such as methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, 2-pentyl, isopentyl, neopentyl, hexyl, 2- hexyl, 3-hexyl and 3-methylpentyl. "Co-Cnalkyl" refers to a single covalent bond (C0) or an alkyl group having from 1 to n carbon atoms; for example "Co-C4alkyl" refers to a single covalent bond or a Ci-C4alkyl group; "C0-C8alkyl" refers to a single covalent bond or a d-C8alkyl group. In some instances, a substituent of an alkyl group is specifically indicated. For example, the term "hydroxyalkyl" refers to an alkyl group substituted with at least one hydroxy substituent {e.g., "C1- C^ydroxyalkyl" refers to a Ci-C4alkyl group that has at least one -OH substituent).
"Alkylene" refers to a divalent alkyl group, as defined above. Co-C4alkylene is a single covalent bond or an alkylene group having 1, 2, 3 or 4 carbon atoms; Cj-Qalkylene is an alkylene group having from 1 to 4 carbon atoms; and CrC6alkylene is an alkylene group having from 1 to 6 carbon atoms.
"Alkenyl" refers to straight or branched chain alkene groups, which comprise at least one unsaturated carbon-carbon double bond. Alkenyl groups include C2-C8alkenyl, C2-C6alkenyl and C2-C4alkenyl groups, which have from 2 to 8, 2 to 6 or 2 to 4 carbon atoms, respectively, such as ethenyl, allyl or isopropenyl. "Alkynyl" refers to straight or branched chain alkyne groups, which have one or more unsaturated carbon-carbon bonds, at least one of which is a triple bond. Alkynyl groups include C2-C8alkynyl, C2-C6alkynyl and C2-C4alkynyl groups, which have from 2 to 8, 2 to 6 or 2 to 4 carbon atoms, respectively.
A "cycloalkyl" is a group that comprises one or more saturated and/or partially saturated rings in which all ring members are carbon, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, adamantyl, decahydro-naphthalenyl, octahydro-indenyl, and partially saturated variants of the foregoing, such as cyclohexenyl. Cycloalkyl groups do not comprise an aromatic ring or a heterocyclic ring. Certain cycloalkyl groups are C3-C8cycloalkyl, in which the group contains a single ring having from 3 to 8 ring members, all of which are carbon. A "(C3-C8cycloalkyl)Co-C4alkyrT is a 3- to 8-membered cycloalkyl group linked via a single covalent bond or a Cj-Qalkylene group.
By "alkoxy," as used herein, is meant an alkyl group as described above attached via an oxygen bridge. Alkoxy groups include Ci-C6alkoxy and CrC4alkoxy groups, which have from 1 to 6 or from 1 to 4 carbon atoms, respectively. Methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, see-butoxy, tert-buXoxy, n-pentoxy, 2-pentoxy, 3-pentoxy, isopentoxy, neopentoxy, hexoxy, 2- hexoxy, 3-hexoxy, and 3-methylρentoxy are representative alkoxy groups.
Similarly, "alkylthio" refers to an alkyl group as described above attached via a sulfur bridge.
The term "oxo," as used herein refers to a keto group (C=O). An oxo group that is a substituent of a nonaromatic carbon atom results in a conversion of -CH2- to -C(=O)-.
Similarly, "imino" refers to a group of the formula C=N. The term "iminoalkyl" refers to an alkyl group as described above substituted with an imine {e.g., a group of the formula
Figure imgf000010_0001
The term "alkanoyl" refers to an acyl group {e.g., -(C=O)-alkyl), in which carbon atoms are in a linear or branched alkyl arrangement and where attachment is through the carbon of the keto group. Alkanoyl groups have the indicated number of carbon atoms, with the carbon of the keto group being included in the numbered carbon atoms. For example a Qalkanoyl group is an acetyl group having the formula -(C=O)CH3. Alkanoyl groups include, for example, C2- Qalkanoyl, C2-C6alkanoyl and C2-C4alkanoyl groups, which have from 2 to 8, from 2 to 6 or from 2 to 4 carbon atoms, respectively. "Ci alkanoyl" refers to -(C=O)H, which (along with C2- Csalkanoyl) is encompassed by the term "Ci-Cgalkanoyl."
An "alkanone" is a ketone group in which carbon atoms are in a linear or branched alkyl arrangement. "C3-C8alkanone," "C3-C6alkanone" and "C3-C4alkanone" refer to an alkanone having from 3 to 8, 6 or 4 carbon atoms, respectively. A C3 alkanone group has the structure -CH2- (C=O)-CH3.
Similarly, "alkyl ether" refers to a linear or branched ether substituent (i.e., an alkyl group that is substituted with an alkoxy group). Alkyl ether groups include C2-C8alkyl ether, C2-C6alkyl ether and C2-C4alkyl ether groups, which have 2 to 8, 6 or 4 carbon atoms, respectively. A C2 alkyl ether has the structure -CH2-O-CH3. The term "alkoxycarbonyl" refers to an alkoxy group attached through a keto (-(C=O)-) bridge (i.e., a group having the general structure -C(=O)-O-alkyl). Alkoxycarbonyl groups include CpC8, Ci-C6 and C]-C4alkoxycarbonyl groups, which have from 1 to 8, 6 or 4 carbon atoms, respectively, in the alkyl portion of the group (i.e., the carbon of the keto bridge is not included in the indicated number of carbon atoms). "Qalkoxycarbonyl" refers to -C(=O)-O-CH3; Qalkoxycarbonyl indicates -C(=O)-O-(CH2)2CH3 or -C(=O)-O-(CH)(CH3)2.
"Alkanoyloxy," as used herein, refers to an alkanoyl group linked via an oxygen bridge (i.e., a group having the general structure -O-C(=O)-alkyl). Alkanoyloxy groups include C2-C8, C2-C6 and C2-C4alkanoyloxy groups, which have from 2 to 8, 6 or 4 carbon atoms, respectively. For example, "C2alkanoyloxy" refers to -O-C(=O)-CH3. Similarly, "alkanoylamino," as used herein, refers to an alkanoyl group linked via a nitrogen bridge (i.e., a group having the general structure -N(R)-C(=O)-alkyl), in which R is hydrogen or Ci-Cβalkyl. Alkanoylamino groups include C2-C8, C2-C6 and C2-C4alkanoylamino groups, which have from 2 to 8, 6 or 4 carbon atoms within the alkanoyl group, respectively.
"Alkylsulfonyl" refers to groups of the formula -(SO2)-alkyl, in which the sulfur atom is the point of attachment. Alkylsulfonyl groups include Ci-Cδalkylsulfonyl and Ci-C4alkylsulfonyl groups, which have from 1 to 6 or from 1 to 4 carbon atoms, respectively. Methylsulfonyl is one representative alkylsulfonyl group. "Ci-C4haloalkylsulfonyl" is an alkylsulfonyl group that has from 1 to 4 carbon atoms and is substituted with at least one halogen (e.g., trifluoromethylsulfonyl) . "Alkylsulfonylamino" refers to groups of the formula -NH-(SO2)-alkyl, in which the nitrogen atom is the point of attachment. Alkylsulfonylamino groups include Ci - C6alkylsulfonylamino and C.1-C4alkylsulfonylaniino groups, which have from 1 to 6 or 1 to 4 carbon atoms, respectively. Methylsulfonylamino is a representative alkylsulfonylamino group.
"Aminosulfonyl" refers to groups of the formula -(SO2)-NH2, in which the sulfur atom is the point of attachment. The term "mono- or di-(CrC8alkyl)aminosulfonyl" refers to groups that satisfy the formula -(SO2)-NR2, in which the sulfur atom is the point of attachment, and in which one R is d-C8alkyl and the other R is hydrogen or an independently chosen Q-Cgalkyl.
"Alkylamino" refers to a secondary or tertiary amine that has the general structure -NH- alkyl or -N(alkyl)(alkyl), wherein each alkyl is selected independently from alkyl, cycloalkyl and
(cycloalkyl)alkyl groups. Such groups include, for example, mono- and di-(Q-C8alkyl)amino groups, in which each CrC8alkyl may be the same or different, as well as mono- and di-(Q-
C6alkyl)amino groups and mono- and di-(Ci-C4alkyl)amino groups.
"Alkylaminoalkyl" refers to an alkylamino group linked via an alkylene group (i.e., a group having the general structure -alkylene-NH-alkyl or -alkylene-N(alkyl)(alkyl)) in which each alkyl is selected independently from alkyl, cycloalkyl and (cycloalkyl)alkyl groups. Alkylaminoalkyl groups include, for example, mono- and di-(Ci-C8alkyl)aminoCi-C8alkyl, mono- and di-(Ci-C6alkyl)aminoCi-C6alkyl and mono- and di-(Ci-C6alkyl)aminoCrC4alkyl. "Mono- or di-(Ci-C6alkyl)aminoCo-C6alkyr' refers to a mono- or di-(Ci-C6alkyl)amino group linked via a single covalent bond or a C]-C6alkylene group. The following are representative alkylaminoalkyl groups:
Figure imgf000012_0001
It will be apparent that the definition of "alkyl" as used in the terms "alkylamino" and "alkylaminoalkyl" differs from the definition of "alkyl" used for all other alkyl-containing groups, in the inclusion of cycloalkyl and (cycloalkyl)alkyl groups (e.g., (C3-C7cycloalkyl)Co-C6alkyl). Similarly, "alkylaminoalkoxy" refers to an alkylamino group linked via an alkoxy group
(i.e., a group having the general structure -O-alkyl-NH-alkyl or -O-alkyl-N(alkyl)(alkyl)) in which each alkyl is selected independently. Such groups include, for example, mono- and di-(Q-
Figure imgf000012_0003
groups, such as
Figure imgf000012_0002
^
The term "aminocarbonyl" refers to an amide group (i.e., -(C=O)NH2). The term "mono- or di-(Ci-C6alkyl)aminocarbonyl" refers to groups of the formula -(C=O)-N(R)2, in which the carbonyl is the point of attachment, one R is CrC6alkyl and the other R is hydrogen or an independently chosen Ci-C6alkyl. "Mono- or di-(C]-C6alkyl)aminocarbonylC0-C4alkyl" refers to such a group linked via a single covalent bond (i.e., mono- or di-(CrC6alkyl)aminocarbonyl) or a C,-C4alkylene group (i.e., -(C0-C4alkyl)-(C=O)N(CrC8alkyl)2).
The term "halogen" refers to fluorine, chlorine, bromine or iodine.
A "haloalkyl" is an alkyl group that is substituted with 1 or more independently chosen halogens (e.g., "Ci-Cghaloalkyl" groups have from 1 to 8 carbon atoms; "Ci-C6haloalkyl" groups have from 1 to 6 carbon atoms). Examples of haloalkyl groups include, but are not limited to, mono-, di- or tri-fiuoromethyl; mono-, di- or tri-chloromethyl; mono-, di-, tri-, tetra- or penta- fluoroethyl; mono-, di-, tri-, tetra- or penta-chloroethyl; and 1,2,2,2-tetrafluoro-l-trifluoromethyl- ethyl. Typical haloalkyl groups are trifluoromethyl and difluoromethyl. The term "haloalkoxy" refers to a haloalkyl group as defined above attached via an oxygen bridge. "Ci-C8haloalkoxy" groups have 1 to 8 carbon atoms.
A dash ("-") that is not between two letters or symbols is used to indicate a point of attachment for a substituent. For example, -CONH2 is attached through the carbon atom.
A "carbocycle" or "carbocyclic group" comprises at least one ring formed entirely by carbon-carbon bonds (referred to herein as a carbocyclic ring), and does not contain a heterocycle. Unless otherwise specified, each ring within a carbocycle may be independently saturated, partially saturated or aromatic, and is optionally substituted as indicated. A carbocycle generally has from 1 to 3 fused, pendant or spiro rings; carbocycles within certain embodiments have one ring or two fused rings. Typically, each ring contains from 3 to 8 ring members (i.e., C3-C8); C5- Cη rings are recited in certain embodiments. Carbocycles comprising fused, pendant or spiro rings typically contain from 9 to 14 ring members. Certain carbocycles are C4-Ci0 (i.e., contain from 4 to 10 ring members, and one or two rings). Certain representative carbocycles are cycloalkyl as described above. Other carbocycles are aryl (i.e., contain at least one aromatic carbocyclic ring, with or without one or more additional aromatic and/or cycloalkyl rings). Such aryl carbocycles include, for example, phenyl, naphthyl (e.g., 1-naphthyl and 2-naphthyl), fluorenyl, indanyl and 1,2,3,4-tetrahydro-naphthyl.
Certain carbocycles recited herein are C6-CiOarylCo-C8alkyl groups (i.e., groups in which a 6- to 10-membered carbocyclic group comprising at least one aromatic ring is linked via a single covalent bond or a Ci-C8alkylene group). Such groups include, for example, phenyl and indanyl, as well as groups in which either of the foregoing is linked via CrC8alkylene, preferably via Cr Qalkylene. Phenyl groups linked via a single covalent bond or Ci-C6alkylene group are designated phenylCo-Cβalkyl (e.g., benzyl, 1-phenyl-ethyl, 1 -phenyl -propyl and 2-phenyl-ethyl).
A "heterocycle" or "heterocyclic group" has from 1 to 3 fused, pendant or spiro rings, at least one of which is a heterocyclic ring (i.e., one or more ring atoms is a heteroatom independently chosen from O, S and N, with the remaining ring atoms being carbon). Additional rings, if present, may be heterocyclic or carbocyclic. Typically, a heterocyclic ring comprises 1, 23 3 or 4 heteroatoms; within certain embodiments each heterocyclic ring has 1 or 2 heteroatoms per ring. Each heterocyclic ring generally contains from 3 to 8 ring members (rings having from 4 or 5 to 7 ring members are recited in certain embodiments) and heterocycles comprising fused, pendant or spiro rings typically contain from 9 to 14 ring members. Certain heterocycles comprise a sulfur atom as a ring member; in certain embodiments, the sulfur atom is oxidized to SO or SO2. Heterocycles may be optionally substituted with a variety of substituents, as indicated. Unless otherwise specified, a heterocycle may be a heterocycloalkyl group (i.e., each ring is saturated or partially saturated) or a heteroaryl group (i.e., at least one ring within the group is aromatic), such as a 5- to 10-membered heteroaryl (which may be monocyclic or bicyclic) or a 6-membered heteroaryl (e.g., pyridyl orpyrimidyl). Heterocyclic groups include, for example, azepanyl, azocinyl, benzimidazolyl, benzimidazolinyl, benzisothiazolyl, benzisoxazolyl, benzofuranyl, benzothiofuranyl, benzoxazolyl, benzothiazolyl, benztetrazolyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, dihydrofuro[2,3-b]tetrahydrofuranyl, dihydroisoquinolinyl, dihydrotetrahydrofuranyl, l,4-dioxa-8- aza-spiro[4.5]decyl, dithiazinyl, furanyl, furazanyl, imidazolinyl, imidazolidinyl, imidazolyl, indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isothiazolyl, isoxazolyl, isoquinolinyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, oxazolidinyl, oxazolyl, phthalazinyl, piperazinyl, piperidinyl, piperidinyl, piperidonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridoimidazolyl, pyridooxazolyl, pyridothiazolyl, pyridyl, pyrimidyl, pyrrolidinyl, pyrrolidonyl, pyrrolinyl, pyrrolyl, quinazolinyl, quinolinyl, quinoxalinyl, quinuclidinyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, thiadiazinyl, thiadiazolyl, thiazolyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thienyl, thiophenyl, thiomoφholinyl and variants thereof in which the sulfur atom is oxidized, triazinyl, and any of the foregoing that are substituted with from 1 to 4 substituents as described above. Certain heterocyclic groups are 4- to 12-membered, 5- to 10-membered, 3- to 7- membered, 4- to 7-membered or 5- to 7-membered groups that contain 1 heterocyclic ring or 2 fused or spiro rings, optionally substituted. 4- to 10-membered heterocycloalkyl groups include, for example, piperidinyl, piperazinyl, pyrrolidinyl, azepanyl, l,4-dioxa-8-aza-spiro[4.5]dec-8-yl, morpholino, thiomorpholino and l,l-dioxo-thiomorpholin-4-yl. Such groups may be substituted as indicated. Representative aromatic heterocycles are azocinyl, pyridyl, pyrimidyl, imidazolyl, tetrazolyl and 3,4-dihydro-lH-isoquinolin-2-yl.
A "heterocycleCo-Cβalkyl" is a heterocyclic group linked via a single covalent bond or Q- Cgalkyl group. For example, a (4- to 7-membered heterocycle)C0-C6alkyl is a heterocyclic group having from 4 to 7 ring members linked via a single covalent bond or an alkyl group having from 1 to 6 carbon atoms. A "(6-membered heteroaryl)C0-C6alkyl" refers to a heteroaryl group linked via a single covalent bond or Ci-C6alkyl group. A "substituent," as used herein, refers to a molecular moiety that is covalently bonded to an atom within a molecule of interest. For example, a ring substituent may be a moiety such as a halogen, alkyl group, haloalkyl group or other group that is covalently bonded to an atom (preferably a carbon or nitrogen atom) that is a ring member. Substituents of aromatic groups are generally covalently bonded to a ring carbon atom. The term "substitution" refers to replacing a hydrogen atom in a molecular structure with a substituent, such that the valence on the designated atom is not exceeded, and such that a chemically stable compound (i.e., a compound that can be isolated, characterized, and tested for biological activity) results from the substitution.
Groups that are "optionally substituted" are unsubstituted or are substituted by other than hydrogen at one or more available positions, typically 1, 2, 3, 4 or 5 positions, by one or more suitable groups (which may be the same or different). Optional substitution is also indicated by the phrase "substituted with from 0 to X substituents," where X is the maximum number of possible substituents. Certain optionally substituted groups are substituted with from 0 to 2, 3 or 4 independently selected substituents (i.e., are unsubstituted or substituted with up to the recited maximum number of substitutents). Other optionally substituted groups are substituted with at least one substituent (e.g., substituted with from 1 to 2, 3 or 4 independently selected substituents).
The terms "VRl" and "capsaicin receptor" are used interchangeably herein to refer to a type 1 vanilloid receptor. Unless otherwise specified, these terms encompass both rat and human
VRl receptors (e.g., GenBank Accession Numbers AF327067, AJ277028 and NM_018727; sequences of certain human VRl cDNAs and the encoded amino acid sequences are provided in
U.S. Patent No. 6,482,611), as well as homologues thereof found in other species.
A "VRl modulator," also referred to herein as a "modulator," is a compound that modulates VRl activation and/or VRl-mediated signal transduction. VRl modulators specifically provided herein are compounds of Formula I and pharmaceutically acceptable salts thereof. Certain preferred VRl modulators are not vanilloids. A VRl modulator may be a VRl agonist or antagonist. Certain modulators bind to VRl with a K; that is less than 1 micromolar, preferably less than 500 nanomolar, 100 nanomolar, 10 nanomolar or 1 nanomolar. A representative assay for determining K; at VRl is provided in Example 5, herein.
A modulator is considered an "antagonist" if it detectably inhibits vanilloid ligand binding to VRl and/or VRl-mediated signal transduction (using, for example, the representative assay provided in Example 6); in general, such an antagonist inhibits VRl activation with a ICS0 value of less than 1 micromolar, preferably less than 500 nanomolar, and more preferably less than 100 nanomolar, 10 nanomolar or 1 nanomolar within the assay provided in Example 6. VRl antagonists include neutral antagonists and inverse agonists. An "inverse agonist" of VRl is a compound that reduces the activity of VRl below its basal activity level in the absence of added vanilloid ligand. Inverse agonists of VRl may also inhibit the activity of vanilloid ligand at VRl and/or binding of vanilloid ligand to VRl. The basal activity of VRl, as well as the reduction in VRl activity due to the presence of VRl antagonist, may be determined from a calcium mobilization assay, such as the assay of Example 6.
A "neutral antagonist" of VRl is a compound that inhibits the activity of vanilloid ligand at VRl, but does not significantly change the basal activity of the receptor (i.e., within a calcium mobilization assay as described in Example 6 performed in the absence of vanilloid ligand, VRl activity is reduced by no more than 10%, preferably by no more than 5%, and more preferably by no more than 2%; most preferably, there is no detectable reduction in activity). Neutral antagonists of VRl may inhibit the binding of vanilloid ligand to VRl.
As used herein a "capsaicin receptor agonist" or "VRl agonist" is a compound that elevates the activity of the receptor above the basal activity level of the receptor (i.e., enhances VRl activation and/or VRl -mediated signal transduction). Capsaicin receptor agonist activity may be identified using the representative assay provided in Example 6. In general, such an agonist has an EC50 value of less than 1 micromolar, preferably less than 500 nanomolar, and more preferably less than 100 nanomolar or 10 nanomolar within the assay provided in Example 6. A "vanilloid" any compound that comprises a phenyl ring with two oxygen atoms bound to adjacent ring carbon atoms (one of which carbon atom is located para to the point of attachment of a third moiety that is bound to the phenyl ring). Capsaicin is a representative vanilloid. A "vanilloid ligand" is a vanilloid that binds to VRl with a K; (determined as described herein) that is no greater than 10 μM. Vanilloid ligand agonists include capsaicin, olvanil, N-arachidonoyl- dopamine and resiniferatoxin (RTX). Vanilloid ligand antagonists include capsazepine and iodo- resiniferatoxin.
A "therapeutically effective amount" (or dose) is an amount that, upon administration to a patient, results in a discernible patient benefit (e.g., provides detectable relief from at least one condition being treated). Such relief may be detected using any appropriate criteria, including alleviation of one or more symptoms such as pain. A therapeutically effective amount or dose generally results in a concentration of compound in a body fluid (such as blood, plasma, serum, CSF, synovial fluid, lymph, cellular interstitial fluid, tears or urine) that is sufficient to alter the binding of vanilloid ligand to VRl in vitro (using the assay provided in Example 5) and/or VRl- mediated signal transduction (using an assay provided in Example 6). It will be apparent that the discernible patient benefit may be apparent after administration of a single dose, or may become apparent following repeated administration of the therapeutically effective dose according to a predetermined regimen, depending upon the indication for which the compound is administered.
By "statistically significant," as used herein, is meant results varying from control at the p<0.1 level of significance as measured using a standard parametric assay of statistical significance such as a student's T test.
A "patient" is any individual treated with a compound provided herein. Patients include humans, as well as other animals such as companion animals (e.g., dogs and cats) and livestock. Patients may be experiencing one or more symptoms of a condition responsive to capsaicin receptor modulation {e.g., pain, exposure to vanilloid ligand, itch, urinary incontinence, overactive bladder, respiratory disorders, cough and/or hiccup), or may be free of such symptom(s) {i.e., treatment may be prophylactic in a patient considered at risk for the development of such symptoms).
HETEROARYL SUBSTITUTED PIPERAZINYL-PYRIDINE ANALOGUES
Within certain aspects, as noted above, the present invention provides heteroaryl substituted piperazinyl-pyridine analogues that may be used in a variety of contexts, including in the treatment of pain {e.g., neuropathic or peripheral nerve-mediated pain); exposure to capsaicin; exposure to acid, heat, light, tear gas, air pollutants (such as, for example, tobacco smoke), infectious agents (including viruses, bacteria and yeast), pepper spray or related agents; respiratory conditions such as asthma or chronic obstructive pulmonary disease; itch; urinary incontinence or overactive bladder; cough or hiccup; and/or obesity. Such compounds may also be used within in vitro assays {e.g., assays for receptor activity), as probes for detection and localization of VRl and as standards in ligand binding and VRl -mediated signal transduction assays.
Within certain embodiments, compounds of Formula I herein further satisfy Formula II:
Formula H
Figure imgf000017_0001
or a pharmaceutically acceptable salt thereof. Within Formula II:
D represents a 5-membered heteroaryl with a nitrogen atom located adjacent to the point of attachment; D, E and G are independently O, S, N, NRla or CRj3; Each Ria is independently chosen from:
(a) hydrogen, halogen, cyano and nitro;
(b) groups of the formula -Q-M-Ry; and
(c) groups that are taken together with an adjacent RJa to form a fused 5- to 7-membered carbocyclic or heterocyclic ring that is optionally substituted with from 1 to 4 substituents independently chosen from halogen, cyano, nitro and groups of the formula -Q-M-Ry; and the remaining variables are as described above for Formula I.
In certain embodiments, compounds provided herein satisfy one or more of Formulas IIa — lid, in which variables are as indicated for Formula II, except as defined below:
Figure imgf000018_0001
Formula IIa Formula lib
Figure imgf000018_0002
Formula Hc Formula Hd
Ria Vγ
Figure imgf000018_0003
Formula He Formula Hf
Within Formulas Hc and Ed, D is O or S; and in Formulas lie and Hf, E is N, O or S.
Within certain embodiments of the above Formulas, variables are as follows:
Ar i, R1 and Rla
Certain Ar1 groups satisfy the formula:
Figure imgf000018_0004
. Representative such groups include, for example:
Figure imgf000018_0005
Figure imgf000018_0006
; in which Ria is as described above. In certain embodiments, Rla in the above groups is not hydrogen.
Certain substituents satisfy the formula Q-M-Ry. It will be apparent that if Q is C0 and M is a single covalent bond, then Ry is directly linked (via a single covalent bond) to the Ar1 core ring.
Within certain compounds, Ria is hydrogen, halogen, amino, cyano, CrC6alkyl, Ci- C6haloalkyl, Ci-C6alkoxy, C]-C6alkylsulfonyl, or mono- or di-(Ci-C6alkyl)aminosulfonyl. Representative Ri a groups include, for example, hydrogen, halogen, nitro, cyano, methyl, and trifluoromethyl.
Ar2, R2 and R2a
In certain compounds of the above Formulas, Ar2 is optionally substituted phenyl, pyridyl {i.e., 2-pyridyl, 3-pyridyl or 4-ρyridyl) or pyrimidyl. In other compounds, Ar2 is a 9- to 12- membered bicyclic aryl or heteroaryl group that is optionally substituted as described above. Preferably, Ar2 is substituted with from 0 to 3 or from 1 to 3 substituents independently chosen from R2 as described above. In certain compounds, Ar2 has at least one substituent (R2) and each R2 is independently chosen from amino, cyano, halogen, -SO2NH2, Ci-C4alkyl, Q-C4haloalkyl, Q- C4hydroxyalkyl, Q-C4alkoxy, Q-Qalkylthio, Ci-C4haloalkoxy5 mono- and di-(Q- C4alkyl)aminoCo-C4alkyl, C2-C4alkanoyl, C1-C4alkoxycarbonyl, d-C4alkylsulfonyl Q- C4haloalkylsulfonyl and mono- and di-(Ci-Qalkyl)aminosulfonyl. More preferably, the substituents of Ar2 are independently chosen from amino, cyano, halogen, Q-C4alkyl, Q- Qhaloalkyl, Q-C4alkoxy, Ci-C4haloalkoxy, Ci-Qalkylthio and mono- and di-(Q- C4alkyl)aminoCo-Qalkyl. In certain such compounds, Ar2 is substituted meta and/or para to the point of attachment, wherein the point of attachment refers to the attachment to the core ring. In other words, if Ar2 is phenyl, the phenyl is mono-substituted at the 3 -position, mono-substituted at the 4-position, or di-substituted and the 3- and 4-positions. Preferred Ar2 groups include phenyl, pyridyl and pyrimidyl, each of which is substituted with from 0 to 3 or from 1 to 3 substituents as described herein.
Within certain Ar2 groups, one R2 is taken together with an adjacent R2 to form a fused carbocycle or heterocycle. Representative such groups include, for example, the following bicyclic groups, optionally substituted as described herein:
Figure imgf000019_0002
Figure imgf000019_0001
ell as variants of the foregoing in which the fused ring contains one or more additional double bonds, such as:
Figure imgf000020_0001
The variable R2a, when present, is generally as described above; in certain embodiments, each R2a is independently chosen from amino, cyano, halogen, -SO2NH2, -COOH, Ci-C4alkyl, C1- C4haloalkyl, Ci-C4hydroxyalkyl, Ci-C4alkoxy, CrC4alkylthio, CrC4haloalkoxy, mono- and di- (Ci-C4alkyl)aminoC0-C4alkyl, C2-C4alkanoyl, C]-C4alkoxycarbonyl, Ci-C4alkylsulfonyl, Q- C4haloalkylsulfonyl and mono- and di-(Ci-C4alkyl)sulfonamido. In further such compounds, A is CH or CR2a, and each R2a is independently chosen from cyano, halogen, Q-^alkyl, Cr C4haloalkyl, Ci-C4alkoxy and mono- and di-(CrC4alkyl)aminoC0-C2alkyl. In other such compounds, at least one of A, B, E and T is N. Certain Ar2 groups have the formula:
Figure imgf000020_0002
wherein B and E are as described above.
Rs
In the definition Of R3, the variable "L" is defined as Co-C6alkylene or Ci-C6alkylene that is taken together with R5, R6 or R7 to form a 4- to 7-membered heterocycle. In any heterocycle so formed, at least one carbon atom present in L is also a ring atom, and is covalently bonded to a component atom OfR5, R6 or R7. The resulting heterocycle may be a heterocycloalkyl group (e.g., tetrahydrofuranyl, morpholinyl, piperidinyl or piperazinyl) or a heteroaryl group, such as pyridyl, pyrimidyl or tetrahydrofuranyl. R3 groups comprising such a heterocycle include, for example:
>V NH
W 1^ and
R3, in certain embodiments of the above Formulas, is a group of the formula:
Λ-N \- wherein:
L is C0-C3alkyl; and R5 and R6 are:
(a) independently chosen from hydrogen, Ci-C6alkyl, C2-C6alkenyl, (C3-C3cycloalkyl)Co- C4alkyl, C2-C4alkanoyl and groups that are joined to L to form a 4- to 7-membered heterocycle; or
(b) joined to form a 4- to 12-membered heterocycloalkyl; each of which alkyl, alkenyl, (cycloalkyl)alkyl, alkanoyl and heterocycloalkyl is substituted with from 0 to 4 substituents independently chosen from (i) halogen, hydroxy, amino, aminocarbonyl, oxo, -COOH and -SO2NH2; and (ii) CrC4alkyl, C5-C7cycloalkyl, Ci- C4alkoxy, C2-C4alkanoyl, Ci-C4haloalkyl, mono- and di-(Ci-C4alkyl)ammoC0-C2alkyl, mono- 5 and di-(Ci-C4alkyl)aminocarbonylC0-C2alkyl, phenylC0-C4alkyl and (4- to 7-membered heterocycle)C0-C2alkyl, each of which is substituted with from O to 4 secondary substituents independently chosen from halogen, hydroxy, cyano, CrC4alkyl, Ci-C4alkoxy and Cr Qhaloalkyl;
Such R3 groups include, for example, mono- and di-(Ci-C4alkyl)ammo groups that are
10 substituted with from 0 to 4 substituents independently chosen from halogen, hydroxy, amino, oxo, aminocarbonyl, -COOH, -SO2NH2, CrC4alkyl, C2-C4alkenyl, C5-C7cycloalkyl, Ci-C4haloalkyl,
Ci-C4alkoxy, C2-C4alkyl ether, C2-C4alkanoyl, Ci-C4alkylsulfonyl, C2-C4alkanoylamino and mono-
Figure imgf000021_0001
Other R3 groups include phenyl and 4- to 7-membered heterocycles, each of which is
15 substituted with from O to 4 substituents independently chosen from (a) halogen, hydroxy, amino, oxo, aminocarbonyl, -SO2NH2 and -COOH; and (b) Q-Qalkyl, Ci-C4haloalkyl, C2-C4alkenyl,
(C5-C7cycloalkyl)Co-C2alkyl, CrC4alkoxy, C2-C4alkyl ether, C2-C4alkanoyl, Q-Qalkylsulfonyl,
C2-C4alkanoylamino, mono- and di-(Ci-C4alkyl)amino, mono- and di-(Ci-C4alkyl)aminocarbonyl, mono- or di-(Ci-C6alkyl)ammosulfonyl, phenylCo-C4alkyl and (4- to 7-membered heterocycle)C<r
20 C4alkyl, each of which is substituted with from O to 4 secondary substituents independently chosen from halogen, hydroxy, cyano, -COOH, Ci-C4alkyl and Ci-C4haloalkyl. Certain such R3 groups include azetidinyl, pyrrolidinyl, morpholinyl, thiomorpholinyl, piperidinyl, piperazinyl, tetrahydropyridyl and azepanyl, each of which is substituted with from O to 4 substituents independently chosen from: (a) halogen, hydroxy, amino, oxo, aminocarbonyl, -SO2NH2 and -
25 COOH; and (b) CrC4alkyl, C2-Qalkenyl, C3-C7cycloalkyl, CrC4haloalkyl, CrC4alkoxy, C2-
C4alkyl ether, C2-C4alkanoyl, Ci-C4alkylsulfonyl, C2-C4alkanoylamino and mono- and di-(Ci-
C4alkyl)amino, each of which is substituted with from O to 4 secondary substituents independently chosen from hydroxy and halogen.
Representative examples of such R3 groups include the heterocycles:
Figure imgf000021_0002
and substituted heterocycles, such as:
Figure imgf000022_0001
Other such R3 groups are phenyl, pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, imidazolyl, thienyl, oxazolyl or tetrahydrofuranyl, each of which is substituted with from 0 to 4 substituents independently chosen from halogen, hydroxy, amino, aminocarbonyl, -SO2NH2, -COOH, Cj- C4alkyl, C5-C7cycloalkyl, C2-C4alkyl ether, Ci-C4alkoxy, C2-C4alkanoyl, Ci-C4haloalkyl and mono- and di-(Ci-C4alkyl)amino. In certain embodiments, R3 is not -NH2. In other words, if L is a single covalent bond then at least one of R5 and R6 is not hydrogen.
In further embodiments, R3 is
Figure imgf000022_0002
7. In certain such compounds, L is C0-C3alkyl; and R7 is Ci-C6alkyl, C2-C6alkenyl, (C5-C7cycloalkyl)Co-C4alkyl, C2-C4alkanoyl, phenylC0-C6alkyl or (6-membered heteroaryl)C0-C4alkyl, each of which is substituted with from O to 4 substituents independently chosen from halogen, hydroxy, amino, aminocarbonyl, -SO2NH2, -COOH, Q- C4alkyl, C5-C7cycloalkyl, C2-C4alkyl ether, CrC4alkoxy, C2-C4alkanoyl, Ci-C4haloalkyl and mono- and di-(C1-C4alkyl)amino. Such R3 groups include, for example, benzyloxy and Ci- Cgalkoxy, each of which is optionally substituted with halogen, methyl, methoxy or trifluoromethyl.
Within still further embodiments, R3 is a hydrogen, Ci-C6alkyl or a halogen.
Within further compounds of the above Formulas, R3 is Ci-C4alkyl, C3-Cycycloalkyl or Q- C4haloalkyl, each of which is substituted with from O to 4 substituents independently chosen from halogen, hydroxy, cyano, oxo, aminocarbonyl, -SO2NH2, -COOH, C3-C7cycloalkyl, phenyl and 4- to 7-membered heterocycle.
R4
R4, in certain compounds provided herein, represents zero substituents or one methyl, ethyl or oxo group, preferably located at the 2-position of the piperazine. The carbon to which a methyl or ethyl group is attached is chiral in certain embodiments. For example, the group:
Figure imgf000023_0001
is , , or in certain compounds. In other compounds, R4 represents a single oxo substituent.
X, Y and Z
X, Y and Z, as noted above, are independently CRx or N, such that at least one of X, Y and Z is N. In certain embodiments, each Rx is independently chosen from hydrogen and methyl, or each Rx is hydrogen. In certain representative compounds, Z is N (e.g., X and Y are CH). In other compounds provided herein, X is N (e.g., Y and Z are CH). In further compounds Z and X are N,
X and Y are N or Z and Y are N. In further compounds, X, Y and Z are each N.
Certain compounds provided herein satisfy Formula III:
Figure imgf000023_0002
Within Formula IE, variables are as described above. In certain embodiments: D, E and G are independently O, S, N, NRla or CRla; Each Ru is independently chosen from:
(a) hydrogen, halogen, cyano and nitro; and (b) groups of the formula -Q-M-Ry;
Ar2 is phenyl, pyridyl or pyrimidyl, each of which is substituted with from 0 to 3 substituents independently chosen from amino, cyano, halogen, C]-C4alkyl, Ci-C4haloalkyl, Ci-C4alkoxy, C]-C4haloalkoxy, Ci-C4alkylthio, and mono- and di-(Ci-C4alkyl)aminoC0-C4alkyl; and R4 represents 0 substituents or one methyl substituent. Representative compounds provided herein include, but are not limited to, those specifically described in Examples 1-3. It will be apparent that the specific compounds recited herein are representative only, and are not intended to limit the scope of the present invention. Further, as noted above, all compounds provided herein may be present as a free acid or base, or as a pharmaceutically acceptable salt. In addition, other forms such as hydrates and prodrugs of such compounds are specifically contemplated by the present invention.
Within certain aspects, heteroaryl substituted piperazinyl-pyridine analogues provided herein detectably alter (modulate) VRl activity, as determined using an in vitro VRl functional assay such as a calcium mobilization assay. As an initial screen for such activity, a VRl ligand binding assay may be used. References herein to a "VRl ligand binding assay" are intended to refer to a standard in vitro receptor binding assay such as that provided in Example 5, and a "calcium mobilization assay" (also referred to herein as a "signal transduction assay") may be performed as described in Example 6. Briefly, to assess binding to VRl, a competition assay may be performed in which a VRl preparation is incubated with labeled (e.g., 125I or 3H) compound that binds to VRl (e.g., a capsaicin receptor agonist such as RTX) and unlabeled test compound. Within the assays provided herein, the VRl used is preferably mammalian VRl, more preferably human or rat VRl. The receptor may be recombinantly expressed or naturally expressed. The VRl preparation may be, for example, a membrane preparation from HEK293 or CHO cells that recombinantly express human VRl. Incubation with a compound that detectably modulates vanilloid ligand binding to VRl results in a decrease or increase in the amount of label bound to the VRl preparation, relative to the amount of label bound in the absence of the compound. This decrease or increase may be used to determine the Kj at VRl as described herein. In general, compounds that decrease the amount of label bound to the VRl preparation within such an assay are preferred.
Certain VRl modulators provided herein detectably modulate VRl activity at nanomolar (i.e., submicromolar) concentrations, at subnanomolar concentrations, or at concentrations below 100 picomolar, 20 picomolar, 10 picomolar or 5 picomolar.
As noted above, compounds that are VRl antagonists are preferred within certain embodiments. IC50 values for such compounds may be determined using a standard in vitro VRl- mediated calcium mobilization assay, as provided in Example 6. Briefly, cells expressing capsaicin receptor are contacted with a compound of interest and with an indicator of intracellular calcium concentration (e.g., a membrane permeable calcium sensitivity dye such as Fluo-3 or Fura-2 (Molecular Probes, Eugene, OR), each of which produce a fluorescent signal when bound to Ca+4). Such contact is preferably carried out by one or more incubations of the cells in buffer or culture medium comprising either or both of the compound and the indicator in solution. Contact is maintained for an amount of time sufficient to allow the dye to enter the cells (e.g., 1-2 hours). Cells are washed or filtered to remove excess dye and are then contacted with a vanilloid receptor agonist (e.g., capsaicin, RTX or olvanil), typically at a concentration equal to the EC50 concentration, and a fluorescence response is measured. When agonist-contacted cells are contacted with a compound that is a VRl antagonist the fluorescence response is generally reduced by at least 20%, preferably at least 50% and more preferably at least 80%, as compared to cells that are contacted with the agonist in the absence of test compound. The IC50 for VRl antagonists provided herein is preferably less than 1 micromolar, less than 100 nM, less than 10 nM or less than 1 nM. In certain embodiments, VRl antagonists provided herein exhibit no detectable agonist activity an in vitro assay of capsaicin receptor agonism at a concentration of compound equal to the IC50. Certain such antagonists exhibit no detectable agonist activity an in vitro assay of capsaicin receptor agonism at a concentration of compound that is 100-fold higher than the IC50. In other embodiments, compounds that are capsaicin receptor agonists are preferred. Capsaicin receptor agonist activity may generally be determined as described in Example 6. When cells are contacted with 1 micromolar of a compound that is a VRl agonist, the fluorescence response is generally increased by an amount that is at least 30% of the increase observed when cells are contacted with 100 nM capsaicin. The EC50 for VRl agonists provided herein is preferably less than 1 micromolar, less than 100 nM or less than 10 nM.
VRl modulating activity may also, or alternatively, be assessed using a cultured dorsal root ganglion assay as provided in Example 7 and/or an in vivo pain relief assay as provided in Example 8. VRl modulators provided herein preferably have a statistically significant specific effect on VRl activity within one or more functional assays provided herein.
Within certain embodiments, VRl modulators provided herein do not substantially modulate ligand binding to other cell surface receptors, such as EGF receptor tyrosine kinase or the nicotinic acetylcholine receptor. In other words, such modulators do not substantially inhibit activity of a cell surface receptor such as the human epidermal growth factor (EGF) receptor tyrosine kinase or the nicotinic acetylcholine receptor (e.g., the IC50 or IC40 at such a receptor is preferably greater than 1 micromolar, and most preferably greater than 10 micromolar). Preferably, a modulator does not detectably inhibit EGF receptor activity or nicotinic acetylcholine receptor activity at a concentration of 0.5 micromolar, 1 micromolar or more preferably 10 micromolar. Assays for determining cell surface receptor activity are commercially available, and include the tyrosine kinase assay kits available from Panvera (Madison, WI).
In certain embodiments, preferred VRl modulators are non-sedating. In other words, a dose of VRl modulator that is twice the minimum dose sufficient to provide analgesia in an animal model for determining pain relief (such as a model provided in Example 8, herein) causes only transient (i.e., lasting for no more than 1A the time that pain relief lasts) or preferably no statistically significant sedation in an animal model assay of sedation (using the method described by Fitzgerald et al. (1988) Toxicology 49(2-3):433-9). Preferably, a dose that is five times the minimum dose sufficient to provide analgesia does not produce statistically significant sedation. More preferably, a VRl modulator provided herein does not produce sedation at intravenous doses of less than 25 mg/kg (preferably less than 10 mg/kg) or at oral doses of less than 140 mg/kg (preferably less than 50 mg/kg, more preferably less than 30 mg/kg).
If desired, compounds provided herein may be evaluated for certain pharmacological properties including, but not limited to, oral bioavailability (preferred compounds are orally bioavailable to an extent allowing for therapeutically effective concentrations of the compound to be achieved at oral doses of less than 140 mg/kg, preferably less than 50 mg/kg, more preferably less than 30 mg/kg, even more preferably less than 10 mg/kg, still more preferably less than 1 mg/kg and most preferably less than 0.1 mg/kg), toxicity (a preferred compound is nontoxic when a therapeutically effective amount is administered to a subject), side effects (a preferred compound produces side effects comparable to placebo when a therapeutically effective amount of the compound is administered to a subject), serum protein binding and in vitro and in vivo half-life (a preferred compound exhibits an in vivo half-life allowing for Q.I.D. dosing, preferably T.I.D. dosing, more preferably B.I.D. dosing, and most preferably once-a-day dosing). In addition, differential penetration of the blood brain barrier may be desirable for VRl modulators used to treat pain by modulating CNS VRl activity such that total daily oral doses as described above provide such modulation to a therapeutically effective extent, while low brain levels of VRl modulators used to treat peripheral nerve mediated pain may be preferred (i.e., such doses do not provide brain (e.g., CSF) levels of the compound sufficient to significantly modulate VRl activity). Routine assays that are well known in the art may be used to assess these properties, and identify superior compounds for a particular use. For example, assays used to predict bioavailability include transport across human intestinal cell monolayers, including Caco-2 cell monolayers. Penetration of the blood brain barrier of a compound in humans may be predicted from the brain levels of the compound in laboratory animals given the compound (e.g., intravenously). Serum protein binding may be predicted from albumin binding assays. Compound half-life is inversely proportional to the frequency of dosage of a compound. In vitro half-lives of compounds may be predicted from assays of microsomal half-life as described, for example, within Example 7 of published U.S. Application Number 2005/0070547.
As noted above, preferred compounds provided herein are nontoxic. In general, the term "nontoxic" shall be understood in a relative sense and is intended to refer to any substance that has been approved by the United States Food and Drug Administration ("FDA") for administration to mammals (preferably humans) or, in keeping with established criteria, is susceptible to approval by the FDA for administration to mammals (preferably humans). In addition, a highly preferred nontoxic compound generally satisfies one or more of the following criteria: (1) does not substantially inhibit cellular ATP production; (2) does not significantly prolong heart QT intervals; (3) does not cause substantial liver enlargement, or (4) does not cause substantial release of liver enzymes.
As used herein, a compound that does not substantially inhibit cellular ATP production is a compound that satisfies the criteria set forth in Example 8 of published U.S. Application Number 2005/0070547. In other words, cells treated as described therein with 100 μM of such a compound exhibit ATP levels that are at least 50% of the ATP levels detected in untreated cells. In more highly preferred embodiments, such cells exhibit ATP levels that are at least 80% of the ATP levels detected in untreated cells.
A compound that does not significantly prolong heart QT intervals is a compound that does not result in a statistically significant prolongation of heart QT intervals (as determined by electrocardiography) in guinea pigs, minipigs or dogs upon administration of a dose that yields a serum concentration equal to the EC50 or IC5Q for the compound. In certain preferred embodiments, a dose of 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 40 or 50 mg/kg administered parenterally or orally does not result in a statistically significant prolongation of heart QT intervals.
A compound does not cause substantial liver enlargement if daily treatment of laboratory rodents {e.g., mice or rats) for 5-10 days with a dose that yields a serum concentration equal to the EC50 or IC50 for the compound results in an increase in liver to body weight ratio that is no more than 100% over matched controls. In more highly preferred embodiments, such doses do not cause liver enlargement of more than 75% or 50% over matched controls. If non-rodent mammals (e.g., dogs) are used, such doses should not result in an increase of liver to body weight ratio of more than 50%, preferably not more than 25%, and more preferably not more than 10% over matched untreated controls. Preferred doses within such assays include 0.01, 0.05. 0.1, 0.5, 1, 5, 10, 40 or 50 mg/kg administered parenterally or orally.
Similarly, a compound does not promote substantial release of liver enzymes if administration of twice the minimum dose that yields a serum concentration equal to the EC50 or IC50 at VRl for the compound does not elevate serum levels of ALT, LDH or AST in laboratory animals {e.g., rodents) by more than 100% over matched mock-treated controls. In more highly preferred embodiments, such doses do not elevate such serum levels by more than 75% or 50% over matched controls. Alternatively, a compound does not promote substantial release of liver enzymes if, in an in vitro hepatocyte assay, concentrations (in culture media or other such solutions that are contacted and incubated with hepatocytes in vitro) that are equal to the EC50 or IC50 for the compound do not cause detectable release of any of such liver enzymes into culture medium above baseline levels seen in media from matched mock-treated control cells. In more highly preferred embodiments, there is no detectable release of any of such liver enzymes into culture medium above baseline levels when such compound concentrations are five-fold, and preferably ten-fold the EC5O or IC50 for the compound. In other embodiments, certain preferred compounds do not inhibit or induce microsomal cytochrome P450 enzyme activities, such as CYP1A2 activity, CYP2A6 activity, CYP2C9 activity, CYP2C19 activity, CYP2D6 activity, CYP2E1 activity or CYP3A4 activity at a concentration equal to the EC50 or IC50 at VRl for the compound.
Certain preferred compounds are not clastogenic {e.g., as determined using a mouse erythrocyte precursor cell micronucleus assay, an Ames micronucleus assay, a spiral micronucleus assay or the like) at a concentration equal the EC50 or IC50 for the compound. In other embodiments, certain preferred compounds do not induce sister chromatid exchange {e.g., in Chinese hamster ovary cells) at such concentrations.
For detection purposes, as discussed in more detail below, VRl modulators provided herein may be isotopically-labeled or radiolabeled. For example, compounds may have one or more atoms replaced by an atom of the same element having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be present in the compounds provided herein include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine, such as 2H, 3H, 11C, 13C, 14C, 15N5 18O, 170, 31P, 32P, 35S, 18F and 36Cl. In addition, substitution with heavy isotopes such as deuterium (i.e., 2H) can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances.
PREPARATION OF HETEROARYL SUBSTITUTED PlPERAZINYL-PYRIDINE ANALOGUES
Heteroaryl substituted piperazinyl-pyridine analogues may generally be prepared using standard synthetic methods. Starting materials are commercially available from suppliers such as Sigma-Aldrich Corp. (St. Louis, MO), or may be synthesized from commercially available precursors using established protocols. By way of example, a synthetic route similar to that shown in any of the following Schemes may be used, together with synthetic methods known in the art of synthetic organic chemistry, or variations thereon as appreciated by those skilled in the art. Each variable in the following schemes refers to any group consistent with the description of the compounds provided herein.
In the Schemes that follow, the term "catalyst" refers to a suitable transition metal catalyst such as, but not limited to, tetrakis(triphenylphosphine)palladium(0) or palladium(II) acetate. In addition, the catalytic systems may include ligands such as, but not limited to, 2- (Dicyclohexylphosphino)biphenyl and tri-ter/-butylphosphine, and may also include a base such as K3PO4, Na2CO3 or sodium or potassium tert-hutoxide. Transition metal-catalyzed reactions can be carried out at ambient or elevated temperatures using various inert solvents including, but not limited to, toluene, dioxane, DMF, N-methylpyrrolidinone, ethyleneglycol, dimethyl ether, diglyme and acetonitrile. When used in conjunction with suitable metallo-aryl reagents, transition metal-catalyzed (hetero)aryl/aryl coupling reactions can be used to prepare the compounds encompassed in general structures 5C (Scheme 5), 6-D and 6-E (Scheme 6) and 7-F (Scheme 7). Commonly employed reagent/catalyst pairs include aryl boronic acid/palladium(O) (Suzuki reaction; Miyaura and Suzuki (1995) Chemical Reviews 95:2457) and aryl trialkylstannane/palladium(O) (Stille reaction; T. N. Mitchell, (1992) Synthesis P:803-815), arylzinc/palladium(O) and aryl Grignard/nickel(II). In addition, metal-catalyzed (hetero)aryl/amine coupling reactions (Buchwald-Hartwig cross-coupling reaction; J. F. Hartwig, Angew. Chem. Int. Ed. 37:2046-2067 (1998)) can be used to prepare the compounds encompassed in general structures 4E (Scheme 4), 5E (Scheme 5), and 8F (Scheme 8) and 9H (Scheme 9). Other definitions used in the following Schemes and elsewhere herein are:
BINAP (rαc)-2,2'-Bis(diphenylphosphino)- 1 , 1 '-binaphthyl Boc t-butycarbamoyl
Bu butyl CDCl3 deuterated chloroform δ chemical shift
DCM dichloromethane or methylene chloride
DDQ 2,3-dichloro-5,6-dicyano-l,4-benzoquinone
DBAL diisobutylaluminum hydride
DIEA N,N-diisopropylethylamine
DMA N,N-dimethylacetamide
DMF dimethylformamide
DMSO dimethylsulfoxide
DPPF 1 , 1 '-bis(diphenylphosphino)ferrocene
Et3N triethylamine
EtOAc ethyl acetate
EtOH ethanol
1H NMR proton nuclear magnetic resonance
HOAc acetic acid
HPLC high pressure liquid chromatography
Hz hertz
KOAc potassium acetate
LCMS liquid chromatography/mass spectrometry
MS mass spectrometry
(M+l) mass + 1 m-CPBA wz-chloroperbenzoic acid
MeOH methanol
MsCl methanesulfonyl chloride
NaNHCN sodium cyanamide n-BuLi n-butyl lithium
NIS N-iodosuccinirnide
Tf -SO2CF3
Pd2(dba)3 tris(dibenzylideneacetone)dipalladium(0)
Pd(PPh3)4 tetrakis(triphenylphosphme)palladium(0)
PhNEt2 diethyl-phenyl-amine, also referred to as Ν,Ν-diethylaniline
PPh3 triphenylphosphine t-BuOK potassium /er/-butoxide
THF tetrahydrofuran TLC thin layer chromatography
Scheme 1
Figure imgf000030_0001
Scheme 2
Figure imgf000030_0002
Scheme 3
Figure imgf000031_0001
R-I aX R-I3-X
(X=CI1Br) (X=CI1Br)
Base Base
Figure imgf000031_0002
Scheme 4
Figure imgf000031_0003
4-A 4-B 4-C
Figure imgf000031_0004
Scheme 5
Scheme 6
Figure imgf000032_0002
Ar2-B(OH)2 or Ar2-B(OH)2 or Ar2-Sn(bu)3 Ar2-Sn(bu)3
Figure imgf000032_0003
Figure imgf000033_0001
Scheme 8
Figure imgf000033_0002
Scheme 9
Figure imgf000034_0001
9-B 9-C
Figure imgf000034_0002
Scheme 10
Figure imgf000034_0003
t-BuOK / BINAP Toluene/ 80 C
Figure imgf000034_0004
In certain embodiments, a compound provided herein may contain one or more asymmetric carbon atoms, so that the compound can exist in different stereoisomeric forms. Such forms can be, for example, racemates or optically active forms. As noted above, all stereoisomers are encompassed by the present invention. Nonetheless, it may be desirable to obtain single enantiomers (i.e., optically active forms). Standard methods for preparing single enantiomers include asymmetric synthesis and resolution of the racemates. Resolution of the racemates can be accomplished, for example, by conventional methods such as crystallization in the presence of a resolving agent, or chromatography using, for example a chiral HPLC column. Compounds may be radiolabeled by carrying out their synthesis using precursors comprising at least one atom that is a radioisotope. Each radioisotope is preferably carbon (e.g.,
14C), hydrogen (e.g., 3H)5 sulfur (e.g., 35S), or iodine (e.g., 125I). Tritium labeled compounds may also be prepared catalytically via platinum-catalyzed exchange in tritiated acetic acid, acid- catalyzed exchange in tritiated trifluoroacetic acid, or heterogeneous-catalyzed exchange with tritium gas using the compound as substrate. In addition, certain precursors may be subjected to tritium-halogen exchange with tritium gas, tritium gas reduction of unsaturated bonds, or reduction using sodium borotritide, as appropriate. Preparation of radiolabeled compounds may be conveniently performed by a radioisotope supplier specializing in custom synthesis of radiolabeled probe compounds.
PHARMACEUTICAL COMPOSITIONS
The present invention also provides pharmaceutical compositions comprising one or more compounds provided herein, together with at least one physiologically acceptable carrier or excipient. Pharmaceutical compositions may comprise, for example, one or more of water, buffers (e.g., neutral buffered saline or phosphate buffered saline), ethanol, mineral oil, vegetable oil, dimethylsulfoxide, carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, adjuvants, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione and/or preservatives. In addition, other active ingredients may (but need not) be included in the pharmaceutical compositions provided herein. Pharmaceutical compositions may be formulated for any appropriate manner of administration, including, for example, topical, oral, nasal, rectal or parenteral administration. The term parenteral as used herein includes subcutaneous, intradermal, intravascular (e.g., intravenous), intramuscular, spinal, intracranial, intrathecal and intraperitoneal injection, as well as any similar injection or infusion technique. In certain embodiments, compositions suitable for oral use are preferred. Such compositions include, for example, tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs. Within yet other embodiments, pharmaceutical compositions may be formulated as a lyophilizate. Formulation for topical administration may be preferred for certain conditions (e.g., in the treatment of skin conditions such as burns or itch). Formulation for direct administration into the bladder (intravesicular administration) may be preferred for treatment of urinary incontinence and overactive bladder.
Compositions intended for oral use may further comprise one or more components such as sweetening agents, flavoring agents, coloring agents and/or preserving agents in order to provide appealing and palatable preparations. Tablets contain the active ingredient in admixture with physiologically acceptable excipients that are suitable for the manufacture of tablets. Such excipients include, for example, inert diluents (e.g., calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate), granulating and disintegrating agents (e.g., corn starch or alginic acid), binding agents (e.g., starch, gelatin or acacia) and lubricating agents (e.g., magnesium stearate, stearic acid or talc). The tablets may be uncoated or they may be coated by known techniques. Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent (e.g., calcium carbonate, calcium phosphate or kaolin), or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium (e.g., peanut oil, liquid paraffin or olive oil).
Aqueous suspensions contain the active material(s) in admixture with suitable excipients, such as suspending agents (e.g., sodium carboxymethylcellulose, methylcellulose, hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia); and dispersing or wetting agents (e.g., naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with fatty acids such as polyoxyethylene stearate, condensation products of ethylene oxide with long chain aliphatic alcohols such as heptadecaethyleneoxycetanol, condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides such as polyethylene sorbitan monooleate). Aqueous suspensions may also comprise one or more preservatives, such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and/or one or more sweetening agents, such as sucrose or saccharin.
Oily suspensions may be formulated by suspending the active ingredient(s) in a vegetable oil (e.g., arachis oil, olive oil, sesame oil or coconut oil) or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and/or flavoring agents may be added to provide palatable oral preparations. Such suspensions may be preserved by the addition of an antioxidant such as ascorbic acid.
Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, such as sweetening, flavoring and coloring agents, may also be present.
Pharmaceutical compositions may also be formulated as oil-in-water emulsions. The oily phase may be a vegetable oil (e.g., olive oil or arachis oil), a mineral oil (e.g., liquid paraffin) or a mixture thereof. Suitable emulsifying agents include naturally-occurring gums (e.g., gum acacia or gum tragacanth), naturally-occurring phosphatides (e.g., soy bean lecithin, and esters or partial esters derived from fatty acids and hexitol), anhydrides (e.g., sorbitan monoleate) and condensation products of partial esters derived from fatty acids and hexitol with ethylene oxide (e.g., polyoxyethylene sorbitan monoleate). An emulsion may also comprise one or more sweetening and/or flavoring agents.
Syrups and elixirs may be formulated with sweetening agents, such as glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also comprise one or more demulcents, preservatives, flavoring agents and/or coloring agents.
Formulations for topical administration typically comprise a topical vehicle combined with active agent(s), with or without additional optional components. Suitable topical vehicles and additional components are well known in the art, and it will be apparent that the choice of a vehicle will depend on the particular physical form and mode of delivery. Topical vehicles include water; organic solvents such as alcohols (e.g., ethanol or isopropyl alcohol) or glycerin; glycols {e.g., butylene, isoprene or propylene glycol); aliphatic alcohols (e.g., lanolin); mixtures of water and organic solvents and mixtures of organic solvents such as alcohol and glycerin; lipid-based materials such as fatty acids, acylglycerols (including oils, such as mineral oil, and fats of natural or synthetic origin), phosphoglycerides, sphingolipids and waxes; protein-based materials such as collagen and gelatin; silicone-based materials (both non-volatile and volatile); and hydrocarbon- based materials such as microsponges and polymer matrices. A composition may further include one or more components adapted to improve the stability or effectiveness of the applied formulation, such as stabilizing agents, suspending agents, emulsifying agents, viscosity adjusters, gelling agents, preservatives, antioxidants, skin penetration enhancers, moisturizers and sustained release materials. Examples of such components are described in Martindale~The Extra Pharmacopoeia (Pharmaceutical Press, London 1993) and Remington: TIte Science and Practice of Pharmacy, 21st ed., Lippincott Williams & Wilkins, Philadelphia, PA (2005). Formulations may comprise microcapsules, such as hydroxymethylcellulose or gelatin-microcapsules, liposomes, albumin microspheres, microemulsions, nanoparticles or nanocapsules. A topical formulation may be prepared in any of a variety of physical forms including, for example, solids, pastes, creams, foams, lotions, gels, powders, aqueous liquids and emulsions. The physical appearance and viscosity of such pharmaceutically acceptable forms can be governed by the presence and amount of emulsifier(s) and viscosity adjuster(s) present in the formulation. Solids are generally firm and non-pourable and commonly are formulated as bars or sticks, or in particulate form; solids can be opaque or transparent, and optionally can contain solvents, emulsifiers, moisturizers, emollients, fragrances, dyes/colorants, preservatives and other active ingredients that increase or enhance the efficacy of the final product. Creams and lotions are often similar to one another, differing mainly in their viscosity; both lotions and creams may be opaque, translucent or clear and often contain emulsifiers, solvents, and viscosity adjusting agents, as well as moisturizers, emollients, fragrances, dyes/colorants, preservatives and other active ingredients that increase or enhance the efficacy of the final product. Gels can be prepared with a range of viscosities, from thick or high viscosity to thin or low viscosity. These formulations, like those of lotions and creams, may also contain solvents, emulsifiers, moisturizers, emollients, fragrances, dyes/colorants, preservatives and other active ingredients that increase or enhance the efficacy of the final product. Liquids are thinner than creams, lotions, or gels and often do not contain emulsifiers. Liquid topical products often contain solvents, emulsifiers, moisturizers, emollients, fragrances, dyes/colorants, preservatives and other active ingredients that increase or enhance the efficacy of the final product.
Suitable emulsifiers for use in topical formulations include, but are not limited to, ionic emulsifiers, cetearyl alcohol, non-ionic emulsifiers like polyoxyethylene oleyl ether, PEG-40 stearate, ceteareth-12, ceteareth-20, ceteareth-30, ceteareth alcohol, PEG-100 stearate and glyceryl stearate. Suitable viscosity adjusting agents include, but are not limited to, protective colloids or non-ionic gums such as hydroxyethylcellulose, xanthan gum, magnesium aluminum silicate, silica, microcrystalline wax, beeswax, paraffin, and cetyl palmitate. A gel composition may be formed by the addition of a gelling agent such as chitosan, methyl cellulose, ethyl cellulose, polyvinyl alcohol, polyquaterniums, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carbomer or ammoniated glycyrrhizinate. Suitable surfactants include, but are not limited to, nonionic, amphoteric, ionic and anionic surfactants. For example, one or more of dimethicone copolyol, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, lauramide DEA, cocamide DEA, and cocamide MEA, oleyl betaine, cocamidopropyl phosphatidyl PG-dimonium chloride, and ammonium laureth sulfate may be used within topical formulations. Suitable preservatives include, but are not limited to, antimicrobials such as methylparaben, propylparaben, sorbic acid, benzoic acid, and formaldehyde, as well as physical stabilizers and antioxidants such as vitamin E, sodium ascorbate/ascorbic acid and propyl gallate. Suitable moisturizers include, but are not limited to, lactic acid and other hydroxy acids and their salts, glycerin, propylene glycol, and butylene glycol. Suitable emollients include lanolin alcohol, lanolin, lanolin derivatives, cholesterol, petrolatum, isostearyl neopentanoate and mineral oils. Suitable fragrances and colors include, but are not limited to, FD&C Red No. 40 and FD&C Yellow No. 5. Other suitable additional ingredients that may be included a topical formulation include, but are not limited to, abrasives, absorbents, anti-caking agents, anti-foaming agents, antistatic agents, astringents (e.g., witch hazel, alcohol and herbal extracts such as chamomile extract), binders/excipients, buffering agents, chelating agents, film forming agents, conditioning agents, propellants, opacifying agents, pH adjusters and protectants.
An example of a suitable topical vehicle for formulation of a gel is: hydroxypropylcellulose (2.1%); 70/30 isopropyl alcohol/water (90.9%); propylene glycol (5.1%); and Polysorbate 80 (1.9%). An example of a suitable topical vehicle for formulation as a foam is: cetyl alcohol (1.1%); stearyl alcohol (0.5%; Quaternium 52 (1.0%); propylene glycol (2.0%); Ethanol 95 PGF3 (61.05%); deionized water (30.05%); P75 hydrocarbon propellant (4.30%). All percents are by weight. Typical modes of delivery for topical compositions include application using the fingers; application using a physical applicator such as a cloth, tissue, swab, stick or brush; spraying (including mist, aerosol or foam spraying); dropper application; sprinkling; soaking; and rinsing.
A pharmaceutical composition may be prepared as a sterile injectible aqueous or oleaginous suspension. The compound(s) provided herein, depending on the vehicle and concentration used, can either be suspended or dissolved in the vehicle. Such a composition may be formulated according to the known art using suitable dispersing, wetting agents and/or suspending agents such as those mentioned above. Among the acceptable vehicles and solvents that may be employed are water, 1,3-butanediol, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils may be employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectible compositions, and adjuvants such as local anesthetics, preservatives and/or buffering agents can be dissolved in the vehicle. Pharmaceutical compositions may also be formulated as suppositories (e.g., for rectal administration). Such compositions can be prepared by mixing the drug with a suitable non- irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Suitable excipients include, for example, cocoa butter and polyethylene glycols. Compositions for inhalation typically can be provided in the form of a solution, suspension or emulsion that can be administered as a dry powder or in the form of an aerosol using a conventional propellant (e.g., dichlorodifluoromethane or trichlorofluoromethane).
Pharmaceutical compositions may be formulated as sustained release or controlled-release formulations (i.e., a formulation such as a capsule that effects a slow release of active ingredient(s) following administration). Such formulations may generally be prepared using well known technology and administered by, for example, oral, rectal or subcutaneous implantation, or by implantation at the desired target site. Preferably the formulation provides a relatively constant level of release of active ingredient(s); the release profile can be varied using methods well known in the art, including (a) by varying the thickness or composition of the coating, (b) by altering the amount or manner of addition of plasticizer in the coating, (c) by including additional ingredients, such as release-modifying agents, (d) by altering the composition, particle size or particle shape of the matrix, and (e) by providing one or more passageways through the coating. The amount of modulator contained within a sustained release formulation depends upon, for example, the method of administration (e.g., the site of implantation), the rate and expected duration of release and the nature of the condition to be treated or prevented.
In general, a sustained and/or controlled release formulation comprises a matrix and/or coating that delays disintegration and absorption in the gastrointestinal tract (or implantation site) and thereby provides a delayed action or a sustained action over a longer period. For example, a time delay material such as glyceryl monosterate or glyceryl distearate may be employed. Coatings that regulate release of the modulator include pH-dependent coatings (which may be used to release modulator in the stomach, and enteric coatings (which may be used to release modulator further along the gastrointestinal tract). pH dependent coatings include, for example, shellac, cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropylmethylcellulose phthalate, methacrylic acid ester copolymers and zein.
In addition to or together with the above modes of administration, a compound provided herein may be conveniently added to food or drinking water (e.g., for administration to non-human animals including companion animals (such as dogs and cats) and livestock). Animal feed and drinking water compositions may be formulated so that the animal takes in an appropriate quantity of the composition along with its diet. It may also be convenient to present the composition as a premix for addition to feed or drinking water.
Compounds are generally administered in a therapeutically effective amount. Preferred systemic doses are no higher than 50 mg per kilogram of body weight per day (e.g., ranging from about 0.001 mg to about 50 mg per kilogram of body weight per day), with oral doses generally being about 5-20 fold higher than intravenous doses (e.g., ranging from 0.01 to 40 mg per kilogram of body weight per day).
The amount of active ingredient that may be combined with the carrier materials to produce a single dosage unit will vary depending, for example, upon the patient being treated, the particular mode of administration and any other co-administered drugs. Dosage units generally contain between from about 10 μg to about 500 mg of active ingredient. Optimal dosages may be established using routine testing, and procedures that are well known in the art.
Pharmaceutical compositions may be packaged for treating conditions responsive to VRl modulation (e.g., treatment of exposure to vanilloid ligand or other irritant, pain, itch, obesity or urinary incontinence). Packaged pharmaceutical compositions generally include (i) a container holding a pharmaceutical composition that comprises at least one VRl modulator as described herein and (ii) instructions (e.g., labeling or a package insert) indicating that the contained composition is to be used for treating a condition responsive to VRl modulation in the patient. METHODS OF USE
VRl modulators provided herein may be used to alter activity and/or activation of capsaicin receptors in a variety of contexts, both in vitro and in vivo. Within certain aspects, VRl antagonists may be used to inhibit the binding of vanilloid ligand agonist (such as capsaicin and/or RTX) to capsaicin receptor in vitro or in vivo. In general, such methods comprise the step of contacting a capsaicin receptor with one or more VRl modulators provided herein, in the presence of vanilloid ligand in aqueous solution and under conditions otherwise suitable for binding of the ligand to capsaicin receptor. The VRl modulator(s) are generally present at a concentration that is sufficient to alter the binding of vanilloid ligand to VRl in vitro (using the assay provided in Example 5) and/or VRl -mediated signal transduction (using an assay provided in Example 6). The capsaicin receptor may be present in solution or suspension {e.g., in an isolated membrane or cell preparation), or in a cultured or isolated cell. Within certain embodiments, the capsaicin receptor is expressed by a neuronal cell present in a patient, and the aqueous solution is a body fluid. Preferably, one or more VRl modulators are administered to an animal in an amount such that the VRl modulator is present in at least one body fluid of the animal at a therapeutically effective concentration that is 1 micromolar or less; preferably 500 nanomolar or less; more preferably 100 nanomolar or less, 50 nanomolar or less, 20 nanomolar or less, or 10 nanomolar or less. For example, such compounds may be administered at a therapeutically effective dose that is less than 20 mg/kg body weight, preferably less than 5 mg/kg and, in some instances, less than 1 ∞g/kg.
Also provided herein are methods for modulating, preferably reducing, the signal- transducing activity {i.e., the calcium conductance) of a cellular capsaicin receptor. Such modulation may be achieved by contacting a capsaicin receptor (either in vitro or in vivo) with one or more VRl modulators provided herein under conditions suitable for binding of the modulator(s) to the receptor. The VRl modulator(s) are generally present at a concentration that is sufficient to alter the binding of vanilloid ligand to VRl in vitro and/or VRl -mediated signal transduction as described herein. The receptor may be present in solution or suspension, in a cultured or isolated cell preparation or in a cell within a patient. For example, the cell may be a neuronal cell that is contacted in vivo in an animal. Alternatively, the cell may be an epithelial cell, such as a urinary bladder epithelial cell (urothelial cell) or an airway epithelial cell that is contacted in vivo in an animal. Modulation of signal tranducing activity may be assessed by detecting an effect on calcium ion conductance (also referred to as calcium mobilization or flux). Modulation of signal transducing activity may alternatively be assessed by detecting an alteration of a symptom {e.g., pain, burning sensation, broncho-constriction, inflammation, cough, hiccup, itch, urinary incontinence or overactive bladder) of a patient being treated with one or more VRl modulators provided herein. VRl modulator(s) provided herein are preferably administered to a patient {e.g., a human) orally or topically, and are present within at least one body fluid of the animal while modulating VRl signal-transducing activity. Preferred VRl modulators for use in such methods modulate VRl signal-transducing activity in vitro at a concentration of 1 nanomolar or less, preferably 100 picomolar or less, more preferably 20 picomolar or less, and in vivo at a concentration of 1 micromolar or less, 500 nanomolar or less, or 100 nanomolar or less in a body fluid such as blood. The present invention further provides methods for treating conditions responsive to VRl modulation. Within the context of the present invention, the term "treatment" encompasses both disease-modifying treatment and symptomatic treatment, either of which may be prophylactic (i.e., before the onset of symptoms, in order to prevent, delay or reduce the severity of symptoms) or therapeutic (i.e., after the onset of symptoms, in order to reduce the severity and/or duration of symptoms). A condition is "responsive to VRl modulation" if it is characterized by inappropriate activity of a capsaicin receptor, regardless of the amount of vanilloid ligand present locally, and/or if modulation of capsaicin receptor activity results in alleviation of the condition or a symptom thereof. Such conditions include, for example, symptoms resulting from exposure to VRl- activating stimuli, pain, respiratory disorders (such as cough, asthma, chronic obstructive pulmonary disease, chronic bronchitis, cystic fibrosis and rhinitis, including allergic rhinitis, such as seasonal an perennial rhinitis, and non-allergic rhinitis), depression, itch, urinary incontinence, overactive bladder, hiccup and obesity, as described in more detail below. Such conditions may be diagnosed and monitored using criteria that have been established in the art. Patients may include humans, domesticated companion animals and livestock, with dosages as described above.
Treatment regimens may vary depending on the compound used and the particular condition to be treated; however, for treatment of most disorders, a frequency of administration of 4 times daily or less is preferred. In general, a dosage regimen of 2 times daily is more preferred, with once a day dosing particularly preferred. For the treatment of acute pain, a single dose that rapidly reaches effective concentrations is desirable. It will be understood, however, that the specific dose level and treatment regimen for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy. In general, the use of the minimum dose sufficient to provide effective therapy is preferred. Patients may generally be monitored for therapeutic effectiveness using medical or veterinary criteria suitable for the condition being treated or prevented.
Patients experiencing symptoms resulting from exposure to capsaicin receptor-activating stimuli include individuals with burns caused by heat, light, tear gas or acid and those whose mucous membranes are exposed (e.g., via ingestion, inhalation or eye contact) to capsaicin (e.g., from hot peppers or in pepper spray) or a related irritant such as acid, tear gas, infectious agent(s) or air pollutant(s). The resulting symptoms (which may be treated using VRl modulators, especially antagonists, provided herein) may include, for example, pain, broncho-constriction and inflammation.
Pain that may be treated using the VRl modulators provided herein may be chronic or acute and includes, but is not limited to, peripheral nerve-mediated pain (especially neuropathic pain). Compounds provided herein may be used in the treatment of, for example, postmastectomy pain syndrome, stump pain, phantom limb pain, oral neuropathic pain, toothache (dental pain), denture pain, postherpetic neuralgia, diabetic neuropathy, chemotherapy-induced neuropathy, reflex sympathetic dystrophy, trigeminal neuralgia, osteoarthritis, rheumatoid arthritis, fibromyalgia, Guillain-Barre syndrome, meralgia paresthetica, burning-mouth syndrome and/or pain associated with nerve and root damage, including as pain associated with peripheral nerve disorders {e.g., nerve entrapment and brachial plexus avulsions, amputation, peripheral neuropathies including bilateral peripheral neuropathy, tic douloureux, atypical facial pain, nerve root damage, and arachnoiditis). Additional neuropathic pain conditions include causalgia (reflex sympathetic dystrophy - RSD, secondary to injury of a peripheral nerve), neuritis (including, for example, sciatic neuritis, peripheral neuritis, polyneuritis, optic neuritis, postfebrile neuritis, migrating neuritis, segmental neuritis and Gombault's neuritis), neuronitis, neuralgias {e.g., those mentioned above, cervicobrachial neuralgia, cranial neuralgia, geniculate neuralgia, glossopharyngial neuralgia, migranous neuralgia, idiopathic neuralgia, intercostals neuralgia, mammary neuralgia, mandibular joint neuralgia, Morton's neuralgia, nasociliary neuralgia, occipital neuralgia, red neuralgia, Sluder's neuralgia, splenopalatine neuralgia, supraorbital neuralgia and vidian neuralgia), surgery-related pain, musculoskeletal pain, myofascial pain syndromes, AIDS-related neuropathy, MS-related neuropathy, central nervous system pain {e.g., pain due to brain stem damage, sciatica, and ankylosing spondylitis), and spinal pain, including spinal cord injury-related pain. Headache, including headaches involving peripheral nerve activity may also be treated as described herein. Such pain includes, for example, such as sinus, cluster {i.e., migranous neuralgia) and tension headaches, migraine, temporomandibular pain and maxillary sinus pain. For example, migraine headaches may be prevented by administration of a compound provided herein as soon as a pre-migrainous aura is experienced by the patient. Further conditions that can be treated as described herein include Charcot's pains, intestinal gas pains, ear pain, heart pain, muscle pain, eye pain, orofacial pain {e.g., odontalgia), abdominal pain, gynaecological pain {e.g., menstrual pain, dysmenorrhoea, pain associated with cystitis, labor pain, chronic pelvic pain, chronic prostitis and endometriosis), acute and chronic back pain {e.g., lower back pain), gout, scar pain, hemorrhoidal pain, dyspeptic pains, angina, nerve root pain, "non-painful" neuropathies, complex regional pain syndrome, homotopic pain and heterotopic pain - including pain associated with carcinoma, often referred to as cancer pain {e.g., in patients with bone cancer), pain (and inflammation) associated with venom exposure {e.g., due to snake bite, spider bite, or insect sting) and trauma associated pain {e.g., post-surgical pain, episiotomy pain, pain from cuts, musculoskeletal pain, bruises and broken bones, and burn pain, especially primary hyperalgesia associated therewith). Additional pain conditions that may be treated as described herein include pain associated with respiratory disorders as described above, autoimmune diseases, immunodeficiency disorders, hot flashes, inflammatory bowel disease, gastroesophageal reflux disease (GERD), irritable bowel syndrome and/or inflammatory bowel disease.
Within certain aspects, VRl modulators provided herein may be used for the treatment of mechanical pain. As used herein, the term "mechanical pain" refers to pain other than headache pain that is not neuropathic or a result of exposure to heat, cold or external chemical stimuli. Mechanical pain includes physical trauma (other than thermal or chemical burns or other irritating and/or painful exposures to noxious chemicals) such as post-surgical pain and pain from cuts, bruises and broken bones; toothache; denture pain; nerve root pain; osteoarthritis; rheumatoid arthritis; fibromyalgia; meralgia paresthetica; back pain; cancer-associated pain; angina; carpel tunnel syndrome; and pain resulting from bone fracture, labor, hemorrhoids, intestinal gas, dyspepsia, and menstruation.
Itching conditions that may be treated include psoriatic pruritus, itch due to hemodialysis, aguagenic pruritus, and itching associated with vulvar vestibulitis, contact dermatitis, insect bites and skin allergies. Urinary tract conditions that may be treated as described herein include urinary incontinence (including overflow incontinence, urge incontinence and stress incontinence), as well as overactive or unstable bladder conditions (including bladder detrusor hyper-reflexia, detrusor hyper-reflexia of spinal origin and bladder hypersensitivity), m certain such treatment methods,
VRl modulator is administered via a catheter or similar device, resulting in direct injection of VRl modulator into the bladder. Compounds provided herein may also be used as anti-tussive agents
(to prevent, relieve or suppress coughing) and for the treatment of hiccup, and to promote weight loss in an obese patient.
Within other aspects, VRl modulators provided herein may be used within combination therapy for the treatment of conditions involving pain and/or inflammatory components. Such conditions include, for example, autoimmune disorders and pathologic autoimmune responses known to have an inflammatory component including, but not limited to, arthritis (especially rheumatoid arthritis), psoriasis, Crohn's disease, lupus erythematosus, irritable bowel syndrome, tissue graft rejection, and hyperacute rejection of transplanted organs. Other such conditions include trauma (e.g., injury to the head or spinal cord), cardio- and cerebro-vascular disease and certain infectious diseases.
Within such combination therapy, a VRl modulator is administered to a patient along with an analgesic and/or anti-inflammatory agent. The VRl modulator and analgesic and/or antiinflammatory agent may be present in the same pharmaceutical composition, or may be administered separately in either order. Anti-inflammatory agents include, for example, non- steroidal anti-inflammatory drugs (NSAIDs), non-specific and cyclooxygenase-2 (COX-2) specific cyclooxgenase enzyme inhibitors, gold compounds, corticosteroids, methotrexate, tumor necrosis factor (TNF) receptor antagonists, anti-TNF alpha antibodies, anti-C5 antibodies, and interleukin-1 (IL-I) receptor antagonists. Examples of NSADDs include, but are not limited to ibuprofen (e.g., ADVIL™, MOTRIN™), flurbiprofen (ANSAID™), naproxen or naproxen sodium (e.g., NAPROSYN, ANAPROX, ALEVE™), diclofenac (e.g., CATAFLAM™, VOLTAREN™), combinations of diclofenac sodium and misoprostol (e.g., ARTHROTEC™), sulindac (CUNORπ ™), oxaprozin (DAYPRO™), diflunisal (DOLOBID™), piroxicam (FELDENE™), indomethacin (ESTDOCIN™), etodolac (LODINE™), fenoprofen calcium (NALFON™), ketoprofen (e.g., ORUDIS™, ORUVAIL™), sodium nabumetone (RELAFEN™), sulfasalazine (AZULFIDINE™), tolmetin sodium (TOLECTIN™), and hydroxychloroquine (PLAQUENIL™). One class of NSAIDs consists of compounds that inhibit cyclooxygenase (COX) enzymes; such compounds include celecoxib (CELEBREX™) and rofecoxib (VIOXX™). NSAIDs further include salicylates such as acetylsalicylic acid or aspirin, sodium salicylate, choline and magnesium salicylates (TRILISATE™), and salsalate (DISALCID™), as well as corticosteroids such as cortisone (CORTONE™ acetate), dexamethasone (e.g., DECADRON™), methylprednisolone (MEDROL™), prednisolone (PRELONE™), prednisolone sodium phosphate (PEDIAPRED™), and prednisone (e.g., PREDNICEN-M™, DELTASONE™, STERAPRED™). Further anti-inflammatory agents include meloxicam, rofecoxib, celecoxib, etoricoxib, parecoxib, valdecoxib and tilicoxib.
Suitable dosages for VRl modulator within such combination therapy are generally as described above. Dosages and methods of administration of anti-inflammatory agents can be found, for example, in the manufacturer's instructions in the Physician's Desk Reference. In certain embodiments, the combination administration of a VRl modulator with an anti- inflammatory agent results in a reduction of the dosage of the anti-inflammatory agent required to produce a therapeutic effect (i.e., a decrease in the minimum therapeutically effective amount). Thus, preferably, the dosage of anti-inflammatory agent in a combination or combination treatment method is less than the maximum dose advised by the manufacturer for administration of the antiinflammatory agent without combination administration of a VRl antagonist. More preferably this dosage is less than 3A, even more preferably less than 1A, and highly preferably, less than 1A of the maximum dose, while most preferably the dose is less than 10% of the maximum dose advised by the manufacturer for administration of the anti-inflammatory agent(s) when administered without combination administration of a VRl antagonist. It will be apparent that the dosage amount of VRl antagonist component of the combination needed to achieve the desired effect may similarly be affected by the dosage amount and potency of the anti-inflammatory agent component of the combination.
In certain preferred embodiments, the combination administration of a VRl modulator with an anti-inflammatory agent is accomplished by packaging one or more VRl modulators and one or more anti-inflammatory agents in the same package, either in separate containers within the package or in the same contained as a mixture of one or more VRl antagonists and one or more anti-inflammatory agents. Preferred mixtures are formulated for oral administration (e.g., as pills, capsules, tablets or the like). In certain embodiments, the package comprises a label bearing indicia indicating that the one or more VRl modulators and one or more anti-inflammatory agents are to be taken together for the treatment of an inflammatory pain condition. Within further aspects, VRl modulators provided herein may be used in combination with one or more additional pain relief medications. Certain such medications are also anti- inflammatory agents, and are listed above. Other such medications are analgesic agents, including narcotic agents which typically act at one or more opioid receptor subtypes (e.g., μ, K and/or δ), preferably as agonists or partial agonists. Such agents include opiates, opiate derivatives and opioids, as well as pharmaceutically acceptable salts and hydrates thereof. Specific examples of narcotic analgesics include, within preferred embodiments, alfentanil, alphaprodine, anileridine, bezitramide, buprenorphine, butorphanol, codeine, diacetyldihydromorphine, diacetylmorphine, dihydrocodeine, diphenoxylate, ethylmorphine, fentanyl, heroin, hydrocodone, hydromorphone, isomethadone, levomethorphan, levorphane, levorphanol, meperidine, metazocine, methadone, methorphan, metopon, morphine, nalbuphine, opium extracts, opium fluid extracts, powdered opium, granulated opium, raw opium, tincture of opium, oxycodone, oxymorphone, paregoric, pentazocine, pethidine, phenazocine, piminodine, propoxyphene, racemethorphan, racemorphan, sulfentanyl, thebaine and pharmaceutically acceptable salts and hydrates of the foregoing agents. Other examples of narcotic analgesic agents include acetorphine, acetyldihydrocodeine, acetylmethadol, allylprodine, alphracetylmethadol, alphameprodine, alphamethadol, benzethidine, benzylmorphine, betacetylmethadol, betameprodine, betamethadol, betaprodine, clonitazene, codeine methylbromide, codeine-N-oxide, cyprenorphine, desomorphine, dextromoramide, diampromide, diethylthiambutene, dihydromorphine, dimenoxadol, dimepheptanol, dimethylthiamubutene, dioxaphetyl butyrate, dipipanone, drotebanol, ethanol, ethylmethylthiambutene, etonitazene, etorphine, etoxeridine, furethidine, hydromorphinol, hydroxypethidine, ketobemidone, levonioramide, levophenacylmoφhan, methyldesoφhine, methyldihydromorphme, moφheridine, moφhine methylpromide, moφhine methylsulfonate, moφhine-N-oxide, myrophin, naloxone, naltyhexone, nicocodeine, nicomoφhine, noracymethadol, norlevoφhanol, normethadone, normoφhine, noφipanone, pentazocaine, phenadoxone, phenampromide, phenomoφhan, phenoperidine, piritramide, pholcodine, proheptazoine, properidine, propiran, racemoramide, thebacon, trimeperidine and the pharmaceutically acceptable salts and hydrates thereof.
Further specific representative analgesic agents include, for example acetaminophen (paracetamol); aspirin and other NSAIDs described above; NR2B antagonists; bradykinin antagonists; anti-migraine agents; anticonvulsants such as oxcarbazepine and carbamazepine; antidepressants (such as TCAs, SSRIs, SNRIs, substance P antagonists, etc.); spinal blocks; gabapentin; asthma treatments (such as θ2-adrenergic receptor agonists; leukotriene D4 antagonists (e.g., montelukast); TALWIN® Nx and DEMEROL® (both available from Sanofϊ Winthrop Pharmaceuticals; New York, NY); LEVO-DROMORAN®; BUPRENEX® (Reckitt & Coleman Pharmaceuticals, Inc.; Richmond, VA); MSIR® (Purdue Pharma L.P.; Norwalk, CT); DILAUDID® (Knoll Pharmaceutical Co.; Mount Olive, NJ); SUBLIMAZE®; SUFENTA® (Janssen Pharmaceutica Inc.; Titusville, NJ); PERCOCET®, NUBAIN® and NUMORPHAN® (all available from Endo Pharmaceuticals Inc.; Chadds Ford, PA) HYDROSTAT® IR, MS/S and MS/L (all available from Richwood Pharmaceutical Co. Inc; Florence, KY), ORAMORPH® SR and ROXICODONE® (both available from Roxanne Laboratories; Columbus OH) and STADOL® (Bristol-Myers Squibb; New York, NY). Still further analgesic agents include CB2- receptor agonists, such as AM 1241, and compounds that bind to the o2δ subunit, such as Neurontin (Gabapentin) and pregabalin.
Representative anti-migraine agents for use in combination with a VRl modulator provided herein include CGRP antagonists, ergotamines and 5-HTi agonists, such as sumatripan, naratriptan, zolmatriptan and rizatriptan.
Within still further aspects, VRl modulators provided herein may be used in combination with one or more leukotriene receptor antagonists (e.g., agents that inhibits the cysteinyl leukotriene CySLT1 receptor). CysLTi antagonists include Montelukast (SESfGULAIR®; Merck & Co., Inc.). Such combinations find use in the treatment of pulmonary disorders such as asthma. For the treatment or prevention of cough, a VRl modulator as provided herein may be used in combination with other medication designed to treat this condition, such as antibiotics, anti-inflammatory agents, cystinyl leukotrienes, histamine antagonists, corticosteroids, opioids, NMDA antagonists, proton pump inhibitors, nociceptin, neurokinin (NKl, NK2 and NK3) and bradykinin (BKl and BK2) receptor antagonists, cannabinoids, blockers of Na+-dependent channels and large conductance Ca+2-dependent K+-channel activators. Specific agents include dexbrompheniramine plus pseudoephedrine, loratadine, oxymetazoline, ipratropium, albuterol, beclomethasone, morphine, codeine, pholcodeine and dextromethorphan.
The present invention further provides combination therapy for the treatment of urinary incontinence. Within such aspects, a VRl modulator provided herein may be used in combination with other medication designed to treat this condition, such as estrogen replacement therapy, progesterone congeners, electrical stimulation, calcium channel blockers, antispasmodic agents, cholinergic antagonists, antimuscarinic drugs, tricyclic antidepressants, SNRIs, beta adrenoceptor agonists, phosphodiesterase inhibitors, potassium channel openers, nociceptin/orphanin FQ (OP4) agonists, neurokinin (NKl and NK2) antagonists, P2X3 antagonists, musculotrophic drugs and sacral neuromodulation. Specific agents include oxybutinin, emepronium, tolterodine, flavoxate, flurbiprofen, tolterodine, dicyclomine, propiverine, propantheline, dicyclomine, imipramine, doxepin, duloxetine, l-deamino-8-D-arginine vasopressin, muscarinic receptor antagonists such as Tolterodine (DETROL®; Pharmacia Corporation) and anticholinergic agents such as Oxybutynin (DITROPAN®; Ortho-McNeil Pharmaceutical, Inc., Raritan, NJ). Suitable dosages for VRl modulator within such combination therapy are generally as described above. Dosages and methods of administration of other pain relief medications can be found, for example, in the manufacturer's instructions in the Physician's Desk Reference. In certain embodiments, the combination administration of a VRl modulator with one or more additional pain medications results in a reduction of the dosage of each therapeutic agent required to produce a therapeutic effect (e.g., the dosage or one or both agent may less than 3A, less than V2, less than 1A or less than 10% of the maximum dose listed above or advised by the manufacturer). For use in combination therapy, pharmaceutical compositions as described above may further comprise one or more additional medications as described above. In certain such compositions, the additional medication is an analgesic. Also provided herein are packaged pharmaceutical preparations comprising one or more VRl modulators and one or more additional medications (e.g., analgesics) in the same package. Such packaged pharmaceutical preparations generally include (i) a container holding a pharmaceutical composition that comprises at least one VRl modulator as described herein; (ii) a container holding a pharmaceutical composition that comprises at least one additional medication (such as a pain relief and/or anti-inflammatory medication) as described above and (iii) instructions (e.g., labeling or a package insert) indicating that the compositions are to be used simultaneously, separately or sequentially for treating or preventing a condition responsive to VRl modulation in the patient (such as a condition in which pain and/or inflammation predominates).
Compounds that are VRl agonists may further be used, for example, in crowd control (as a substitute for tear gas) or personal protection (e.g., in a spray formulation) or as pharmaceutical agents for the treatment of pain, itch, urinary incontinence or overactive bladder via capsaicin receptor desensitization. In general, compounds for use in crowd control or personal protection are formulated and used according to conventional tear gas or pepper spray technology.
Within separate aspects, the present invention provides a variety of non-pharmaceutical in vitro and in vivo uses for the compounds provided herein. For example, such compounds may be labeled and used as probes for the detection and localization of capsaicin receptor (in samples such as cell preparations or tissue sections, preparations or fractions thereof). In addition, compounds provided herein that comprise a suitable reactive group (such as an aryl carbonyl, nitro or azide group) may be used in photoaffmity labeling studies of receptor binding sites. In addition, compounds provided herein may be used as positive controls in assays for receptor activity, as standards for determining the ability of a candidate agent to bind to capsaicin receptor, or as radiotracers for positron emission tomography (PET) imaging or for single photon emission computerized tomography (SPECT). Such methods can be used to characterize capsaicin receptors in living subjects. For example, a VRl modulator may be labeled using any of a variety of well known techniques (e.g., radiolabeled with a radionuclide such as tritium, as described herein), and incubated with a sample for a suitable incubation time (e.g., determined by first assaying a time course of binding). Following incubation, unbound compound is removed (e.g., by washing), and bound compound detected using any method suitable for the label employed (e.g., autoradiography or scintillation counting for radiolabeled compounds; spectroscopic methods may be used to detect luminescent groups and fluorescent groups). As a control, a matched sample containing labeled compound and a greater (e.g., 10-fold greater) amount of unlabeled compound may be processed in the same manner. A greater amount of detectable label remaining in the test sample than in the control indicates the presence of capsaicin receptor in the sample. Detection assays, including receptor autoradiography (receptor mapping) of capsaicin receptor in cultured cells or tissue samples may be performed as described by Kuhar in sections 8.1.1 to 8.1.9 of Current Protocols in Pharmacology (1998) John Wiley & Sons, New York.
Compounds provided herein may also be used within a variety of well known cell separation methods. For example, modulators may be linked to the interior surface of a tissue culture plate or other support, for use as affinity ligands for immobilizing and thereby isolating, capsaicin receptors {e.g., isolating receptor-expressing cells) in vitro. Within one preferred embodiment, a modulator linked to a fluorescent marker, such as fluorescein, is contacted with the cells, which are then analyzed (or isolated) by fluorescence activated cell sorting (FACS).
VRl modulators provided herein may further be used within assays for the identification of other agents that bind to capsaicin receptor. In general, such assays are standard competition binding assays, in which bound, labeled VRl modulator is displaced by a test compound. Briefly, such assays are performed by: (a) contacting capsaicin receptor with a radiolabeled VRl modulator as described herein, under conditions that permit binding of the VRl modulator to capsaicin receptor, thereby generating bound, labeled VRl modulator; (b) detecting a signal that corresponds to the amount of bound, labeled VRl modulator in the absence of test agent; (c) contacting the bound, labeled VRl modulator with a test agent; (d) detecting a signal that corresponds to the amount of bound labeled VRl modulator in the presence of test agent; and (e) detecting a decrease in signal detected in step (d), as compared to the signal detected in step (b).
The following Examples are offered by way of illustration and not by way of limitation. Unless otherwise specified all reagents and solvent are of standard commercial grade and are used without further purification. Using routine modifications, the starting materials may be varied and additional steps employed to produce other compounds provided herein.
EXAMPLES Mass spectroscopy data in this and the following Examples is Electrospray MS, obtained in positive ion mode using a Micromass Time-of-Flight LCT (Micromass, Beverly MA), equipped with a Waters 600 pump (Waters Corp., Milford, MA), Waters 996 photodiode array detector, Gilson 215 autosampler (Gilson, Inc. Middleton, WI), and a Gilson 841 microinjector. MassLynx (Advanced Chemistry Development, Inc; Toronto, Canada) version 4.0 software with OpenLynx processing is used for data collection and analysis. MS conditions are as follows: capillary voltage = 3.5 kV; cone voltage = 30 V, desolvation and source temperature = 35O0C and 1200C, respectively; mass range = 181-750 with a scan time of 0.22 seconds and an interscan delay of 0.05 minutes.
Sample volume of 1 microliter is injected onto a 50x4.6mm Chromolith SpeedROD RP- 18e column (Merck KGaA, Darmstadt, Germany), and eluted using a 2-phase linear gradient at 6ml/min flow rate. Sample is detected using total absorbance count over the 220-340nm UV range. The elution conditions are: Mobile Phase A- 95/5/0.05 Water/Methanol/TFA; Mobile Phase B-5/95/0.025 Water/Methanol/TFA. The following gradient is used: Gradient: Timefmin) %B
0 10
0.5 100
1.2 100
1.21 10
Inject to inject cycle 2.2 minutes.
EXAMPLE 1
Preparation of Representative Heteroaryl Substituted Piperazinyl-Pyridine Analogues
This Example illustrates the preparation of representative heteroaryl substituted piperazinyl-pyridine analogues.
A. 4-(4-Fluoro-phenyl)-2-(2-methyl-pyrrolidin-l-ylV6-r4-dH-tetrazol-5-yl)-piperidin-l-yl1 pyrimidine (Compound D
1. Piperidine-4-carbonitrile
HN ^)-CN
Stir 4-cyano-piperidine-l-carboxylic acid tert-butyl ester (Oakwood Products, Inc., 5g, 0.024 mol) in dry dioxane with an excess of 4 N HCl dioxane solution (100 mL). After 2 hours, collect the solid by filtration and wash it with ether (3x). Suspend the solid in toluene and add 14 g of amberlyst bicarbonate resin. Stir overnight, and then filter, washing the resin with additional toluene. Stir the collected resin for an additional 1 hour with a solution of MeOH/ Et3N (4:1). Combine the two organic solutions and concentrate under reduced pressure. Take up in DCM, dry (Na2SO4), and concentrate under reduced pressure to give the freebase product as an oil. 2. 2,4-dichloro-6-(4-fluorophenyl)pyrimidine
Figure imgf000050_0001
Dissolve 4-fluorobromobenzene (8.75 g, 0.05 moles) in anhydrous ether (80 mL) under nitrogen atmosphere and cool to -780C. Add dropwise 1.6 M n-BuLi (34 mL, 0.055 moles) and stir at -780C for 45 minutes. Dissolve 2,4-dichloropyrimidine (7.45 g, 0.05 moles) in Et2O (100 mL), add dropwise to the reaction mixture and warm the reaction mixture to -3O0C. Stir at this temperature for 30 minutes, followed by 00C for 30 minutes. Quench the reaction mixture with acetic acid (3.15 mL, 0.055 moles) and water (0.5 mL, 0.027 moles) dissolved in THF (5.0 mL). Add dropwise a THF (40 mL) solution of DDQ (11.9 g, 0.053 moles) to the reaction mixture. Bring the reaction mixture to room temperature and stir at room temperature for 30 minutes. Cool the reaction mixture to 00C, add 3.0 N aq. NaOH (35 mL) and stir for 30 minutes. Decant the organic layer from the reaction mixture and wash the brown solid with Et2O (3 x 100 mL). Combine the organic layers, wash several times with saturated NaCl solution and dry with MgSO4. Filter and evaporate under vacuum to afford a brown colored solid. Purify the crude by flash column chromatography using 5% EtOAc / hexane to afford the product as white solid.
3. l-[2-Chloro-6-(4-fluoro-phenyl)-pyritnidin-4-yl]-pipe}idine-4-carbonitrile
Figure imgf000051_0001
Stir a mixture of 2,4-dichloro-6-(4-fluorophenyl)pyrimidine (3.6g, 0.015 mol), piperidine-
4-carbonitrile (1.8g, 0.016 mol), and K2CO3 (4.1g, 0.03 mol) in DMA at room temperature for 48 hours. Partition the mixture between EtOAc and brine and separate the layers. Wash the organic layer with 10% NaOH solution (2*) followed by brine. Dry the solution (Na2SO4) and concentrate under reduced pressure to give the crude product. Purify using flash column chromatography with silica gel (30% to 50% EtOAc/ hexanes.
4. l-[6-(4-Fluoroψhenyl)-2-(2~meth)dψyirolidin-l-yl)ψyritnidin-4-yl]-piperidine-4-carbonitήle
Figure imgf000051_0002
Heat a solution of l-[2-chloro-6-(4-fluoro-phenyl)-pyrimidm-4-yl]-piperidine-4- carbonitrile (3.13g, 0.01 mol) and 2-methylpyrrolidine (5 mL, 0.05 mol) in DMA at 1300C for 10 hours. Partition the cooled mixture between EtOAc and brine and separate the layers. Wash the organic layer with 10% NaOH solution (2*) followed by brine. Dry the solution (Na2SO4) and concentrate under reduced pressure to give the crude product. Purify using flash column chromatography with silica gel (30% to 50% EtOAc/ hexanes) to give the product as a foam.
5. 4'(4-Fluoro-phenyl)-2-(2-rnethyl-pyirolidin-l-yl)-6-[4-(lH'tetrazol-5-yl)-piperidin-l-yl] pyrimidine
Figure imgf000052_0001
Heat a mixture of l-[6-(4-fluoro-phenyl)-2-(2-methyl-pyrrolidin-l-yl)-pyrimidin-4-yl]- piperidine-4-carbonitrile (367 mg, 1.0 mmol), sodium azide (72 mg, 1.1 mmol), and ammonium chloride (60 mg, 1.1 mmol) in DMF in a sealed tube at 125°C for 24 hours. Partition the cooled mixture between EtOAc and brine and separate the layers. Wash the organic layer with water (2x) followed by brine. Dry the solution (Na2SO4) and concentrate under reduced pressure to give the crude product. Purify using flash column chromatography with silica gel to give the title product. (M+H) = 409; 1H NMR (400 MHz, CDC13): δ 7.85 (m, 2H), 7.01 (m, 2H), 6.16 (s, IH) 4.35 (m, 3H), 3.60 (m, 2H), 3.0 (m, 4H), 1.55-2.1 (m, 7H), 1.15 (d, 3H). The IC50 determined as described in Example 6 is less than 1 micromolar.
B. 4-(4-Fluoro-phenyl)-6-|"4-(l-methyl-lH-imidazol-2-yl)-piperazin-l-yl1-2-(2-methyl-pyrrolidin- l-yl)-pyrimidine (Compound 2)
1. 2-Chloro-4-(4-fluoro-phenyl)-6-[4-(l-methyl-lH-imidazol-2-yl)-piperazin-l-yl] -pyrimidine
Figure imgf000052_0002
Stir a mixture of 2,4-dichloro-6-(4-fiuorophenyl)pyrimidine (200 mg, 0.82 mmol), 1-(1H- imidazol-2-yl)-piperazine (150 mg, 0.91 mmol; prepared essentially as described in EP 0233051), and K2CO3 (239 mg, 1.7 mmol) in DMA at room temperature for 5 hours. Partition the mixture between EtOAc and 10% NaOH and separate the layers. Wash the organic layer with 10% NaOH (3 x) followed by brine. Dry the solution (Na2SO4) and concentrate under reduced pressure to give the crude product. Purify using preparative plate chromatography (5% MeOH/DCM eluent) to give the product as a clear oil that crystallizes upon standing.
2. 4-(4-Fluoro-phenyl)-6-[4-(l-methyl-lH-imidazol-2-yl)-piperaziiι-l-yl]-2-(2-methyl-pyrrolidin- l-yl)-pyrimidine
Figure imgf000053_0001
Heat a solution of 2-chloro-4-(4-fluoro-phenyl)-6-[4-(l-methyl-lH-imidazol-2-yl)- ρiperazin-l-yl]-ρyrimidine (173 mg, 0.47 mmol) and 2-methylpyrrolidine (237 microliters, 2.33 mmol) in DMA at 1100C for 16 hours. Partition the cooled mixture between EtOAc and 10% NaOH and separate the layers. Wash the organic layer with 10% NaOH solution (2x) followed by brine. Dry the solution (Na2SO4) and concentrate under reduced pressure to give the crude product. Purify using preparative plate silica gel chromatography (2 x 2mm, 10% MeOH/DCM eluent) to yield the title product. Prepare the HCl salt by dissolving in EtOAc and adding excess HCl/ether solution to give the HCl salt as an off-white solid. (M+H) = 424; IH NMR of freebase (300 MHz, CDC13): δ 8.01 (m, 2H), 7.09 (t, 2H), 6.80 (s, IH), 6.70 (s, IH), 4.34 (m, IH)5 3.80 (m, 4H), 3.67 (m, 2H), 3.55 (s, 3H), 3.16 (m, 4H), 2.05 (m, 2H), 1.90 (m, IH), 1.70 (m, IH), 1.31 (d, 3H, J=6.1 Hz). The IC50 determined as described in Example 6 is less than 1 micromolar.
C. 4-(4-Fluoro-phenyl)-6-[4-(l-methyl-lH-imidazol-2-yl)-piperidin-l-vn-2-(2-methyl- pyrrolidin-l-vD-pyrimidine (Compound 3)
/. l-[6-(4-Fluoro-phenyl)-2-(2-methyl-pyrrolidin-l-yl)-pyrimidin-4-yl]-piperidine-4- carbaldehyde
Figure imgf000053_0002
Cool a solution of l-[6-(4-fluoro-phenyl)-2-(2-methyl-pyrrolidin-l-yl)-pyrimidin-4-yl]- piperidine-4-carbonitrile (811 mg, 2.22 mmol) in DCM to -780C using a dry ice acetone bath. Add DIBAL (IM in hexanes) dropwise and stir for 3 hours. Add excess Na2SO4-IO H2O and stir the suspension for 10 minutes before removing the cooling bath. Stir for several hours at room temperature then filter the mixture through celite. Concentrate under reduced pressure and chromatograph the crude product on a silica gel flash column (20% to 30% EtOAc / hexanes eluent) to give the product as a white foam.
2. 4-(4-Fluoro-phenyl)-6-[4-(lH-imidazol-2-yl)-piperidin-l-yl]-2-(2-methyl-pyiTolidin-l-yl)- pyrimidine
Figure imgf000054_0001
Stir a mixture of l-[6-(4-fluoro-phenyl)-2-(2-methyl-pyrrolidin-l-yl)-pyrimidin-4-yl]- piperidine-4-carbaldehyde (134 mg, 0.364 mmol), glyoxal (40% aqueous solution, 106 microliters, 0.727 mmol), and concentrated ammonium hydroxide (~16.5 M, 66 microliters, 1.1 mmol) in MeOH at room temperature for 16 hours. Concentrate the solution under reduced pressure, load onto two 2mm silica gel preparative TLC plates, and elute with 5% MeOH(w/ 2N NH3)ZDCM to afford pure product. (M+H) = 407; 1H NMR (300 MHz, CDC13): δ 7.98 (m, 2H), 7.08 (t, 2H), 6.94 (s, 2H), 6.14 (s, IH), 4.50 (m, 2H), 4.31 (M, IH), 3.65 (m, 2H), 3.00 (m, 4H), 1.6-2.12 (m, 7H), 1.29 (d, 3H5 J = 6.32 Hz).
3. 4-(4'Fluoro-phenyl)-6- [4-(l -methyl- lH-imidazol-2-yl)-pipetidin-l-yl]-2-(2-methyl-pyrrolidin- l-yl)-pyrimidine
Figure imgf000054_0002
To a stirring solution of 4-(4-fluoro-phenyl)-6-[4-(lH-imidazol-2-yl)-piperidin-l-yl]-2-(2- methyl-pvrrolidin-l-yl)-pyrimidine (65 mg, 0.16 mmol) in dry DMF, add NaH as a 60% suspension in mineral oil (~8mg). Stir the mixture at room temperature for 5 minutes and then add 2 drops of MeI. After 30 minutes, partition the mixture between EtOAc and 10% NaOH and separate the layers. Wash the organic layer with 10% NaOH solution (2x) followed by brine. Dry the solution (Na2SO4) and concentrate under reduced pressure to give the crude product. Purify using preparative plate silica gel chromatography to give the title product. (M+H) = 421; 1H NMR (300 MHz, CDC13): δ 7.98 (m, 2H), 7.08 (t, 2H), 6.93 (s, IH), 6.79 (s, IH), 6.26 (s, IH), 4.55 (m, 2H), 4.31 (m, IH), 3.65 (m, 5H), 3.0 (m, 4H) 1.8-2.1 (m, 6H), 1.70 (m, IH), 1.30 (d, 3H, J= 6.3 Hz). The IC50 determined as described in Example 6 is less than 1 micromolar.
EXAMPLE 2 Preparation of Additional Representative Heteroaryl Substituted Piperazinyl-Pyridine Analogues
This Example illustrates the preparation of additional representative heteroaryl substituted piperazinyl-pyridine analogues.
A. 4-(3-Chloro-4-fluoro-phenyl)-6-f4-(4-chloro-ri,2.51thiadiazol-3-yl)-piperazin-l-yll-2-(2- methyl-pyrrolidin-l-yl)-pyrimidine (Compound 4)
/. 2,4-Dichloro-6-(3-chloro-4-fluoro-phenyl)-pyrimidine
Figure imgf000055_0001
This compound is prepared from 2,4-dichloropyrimidine and 4-bromo-2-chloro-l-fluoro- benzene using a procedure analogous to that used for the preparation of 2,4-dichloro-6-(4- fluorophenyl)pyrimidine in Example IA, step 2.
2. 4-[2-Chloro-6-(3-chloro-4-fluoro-phenyl)-pyrimidin-4-yl]-piperazine-l-carboxylic acid tert- butyl ester
Figure imgf000055_0002
Stir a mixture of 2,4-dichloro-6-(4-fluoro-phenyl)-pyrimidine (2.8 g, 11.52 mmol), piperazine-1-carboxylic acid tert-\mty\ ester (12.1 mmol), and K2CO3 (3.2 g, 23.0 mmol) in DMA at room temperature for 16 hours. Dilute with water, extract with EtOAc, and wash with brine. Dry the organic layer (Na2SO4) and concentrate under reduced pressure. Purify the residue by flash column eluting with EtOAc-Hexanes (1:4) to afford 4-[2-chloro-6-(3-chloro-4-fluoro- phenyl)-pyrimidin-4-yl]-piperazine-l-carboxylic acid tert-butyl ester.
3. 4-[6-(3-Chloro-4-fluo7-o-phenyl)-2-(2-methyl-pyrrolidin-l-yl)-pyrimidin-4-yl]-piperazine-l- carboxylic acid tert-butyl ester
Figure imgf000056_0001
Heat a mixture of 4-[2-chloro-6-(3-chloro-4-fluoro-phenyl)-pyrimidin-4-yl]-piperazine-l- carboxylic acid tert-butyl ester (12.3 mmol), 2-methyl-pyrrolidine (16.0 mmol), and K2CO3 (5.2 g, 37.8 mmol) in DMA at 1200C for 16 hours. Dilute with water, extract with EtOAc, and wash with brine. Dry the organic layer (Na2SO4) and concentrate under reduced pressure. Purify the residue by flash column eluting with EtOAc-Hexanes (1:4) to afford the product.
4. 4-(3-Chloro-4-fluoro-phenyl)-2-(2-methyl-py7'rolidin-l-yl)-6-piperazin-l-yl-pyrimidine
Figure imgf000056_0002
Stir 4-[6-(3-chloro-4-fluoro-phenyl)-2-(2-methyl-pyrrolidin-l -yl)-pyrimidin-4-yl]- piperazine-1-carboxylic acid tert-butyl ester (9.88 mmol) in 4M HCl-dioxane (50 mL) for 40 minutes. Concentrate and partition between EtOAc and sat. NaHCO3. Dry the organic layer (Na2SO4) and concentrate under reduced pressure to afford the product. 5. 4-(3-Chloro-4-fluoro-phenyl)-6-[4-(4-chloro-[l,2,5]thiadiazol-3-yl)-piperazin-l-yl]-2-(2- methyl-pyrrolidin-l-yl)-pyrimidine
Figure imgf000057_0001
Heat a mixture of 4-(3-chloro-4-fluoro-phenyl)-2-(2-methyl-pyrrolidin-l-yl)-6-piperazin- 1-yl-pyrimidine (114 mg, 0.303 mmol), 3,4-dichloro-[l,2,5]thiadiazole (0.333 mmol) and diisopropylethylamine (0.33 mmol) in DMSO at 1000C for 16 hours. Partition the cooled mixture between EtOAc and brine and separate the layers. Wash the organic layer with 10% NaOH solution (2x) followed by brine. Dry the solution (Na2SO4) and concentrate under reduced pressure to give the crude product. Purify using flash column chromatography with silica gel (EtOAc/ hexanes eluent) to give the title product. (M+H) = 495; 1H NMR (400 MHz, CDC13): δ
8.08 (d, IH), 7.90 (m, IH), 7.19 (t, IH), 6.24 (s, IH), 4.35 (m, IH), 3.83 (m, 4H), 3.70 (M, 2H),
3.60 (m, 4H), 2.06 (m, 2H), 1.93 (m, IH), 1.70 (m, IH).1.30 (d, 3H, J=6.23 hz). The IC50 determined as described in Example 6 is less than 1 micromolar.
B. 4-r3-Chloro-4-fluoro-phenyl)-6-r4-(l-methyl-lH-imidazol-2-yl')-ϋiperazin-l-vn-2-((R)-2- methyl-pyrrolidin-l-yl)-pyrimidine (Compound 5)
1. 2-Chloro-4-(3-chloro-4-fluoro-phenyl)-6-[4-(l -methyl-1 Η-imidazol-2-yl)-piperazin-l-yl]- pyrimidine
Figure imgf000057_0002
Stir a mixture of 2,4-dichloro-6-(3-chloro-4-fluoro-phenyl)-pyrimidine (211 mg, 0.76 mmol), l-(lH-imidazol-2-yl)-piperazine (139 mg, 0.84 mmol) (prepared essentially as described in EP 0233051), and K2CO3 (222 mg, 1.6 mmol) in DMA at room temperature for 16 hours. Partition the mixture between EtOAc and 10% NaOH and separate the layers. Wash the organic layer with 10% NaOH (3x) followed by brine. Dry the solution (Na2SO4) and concentrate under reduced pressure to give the crude product. Purify using preparative plate chromatography (5% MeOH/DCM eluent).
2. 4-(3-Chloro-4-fluoro-phenyl)-6-[4-(l-methyl-lH4midazol-2-yl)ψiperazin-l-ylJ methyl-pyrrolidin-l-yl)-pyrimidine
Figure imgf000058_0001
Heat a solution of 2-chloro-4-(3-chloro-4-£luoro-phenyl)-6-[4-(l-methyl-lH-imidazol-2- yl)-piperazin-l-yl]-pyrimidine (50 mg, 0.123 mmol) and (R)-2-methylpyrrolidine hydrochloride (prepared essentially as described in US Published Application 2004/0171845; 22 mg, 0.18 mmol) in DMA at 1100C for 16 hours. Partition the cooled mixture between EtOAc and 10% NaOH and separate the layers. Wash the organic layer with 10% NaOH solution (2x) followed by brine. Dry the solution (Na2SO4) and concentrate under reduced pressure to give the crude product. Purify using preparative plate silica gel chromatography (1 x 2mm, 5% MeOH/DCM eluent) to give the title product. Prepare the HCl salt by dissolving in EtOAc and adding excess HCl/ether solution to give the HCl salt as an off-white solid. (M+H) = 456; 1H NMR (300 MHz, CDC13): δ 8.06 (dd, IH), 7.88 (m, IH), 7.26 (s, IH), 7.18 (t, IH), 6.80 (s, IH), 6.70 (s, IH), 6.24 (s, IH), 4.33 (M, IH), 3.80 (m, 4H)5 3.67 (m, 2H), 3.17 (t, 4H), 2.06 (m, 2H), 1.90 (m, IH), 1.70 (m, IH), 1.29 (d, 3H, J=6.3 Hz). The IC50 determined as described in Example 6 is less than 1 micromolar.
C. 4-{4-(3-Chloro-4-fluoro-phenylV6-r4-(l-methyl-lH-imidazol-2-yl)-piperazin-l-yl]-pyridin-2- yll-morpholine (Compound 6) 1. 2,3-dichloro-4-(3-chloro-4-fluoro-phenyl)-pyridine
Figure imgf000058_0002
To a de-gassed mixture of 3-chloro-4-fluorophenylboronic acid (77 mg, 0.44 mmol)), 4- bromo-2,6-dichloro-pyridine (prepared essentially as described in Talik & Plazek (1959) Rocz. Chem. 33:387-392; 100 mg, 0.44 mmol), and 2M Na2CO3 (0.55 mmol), in DME (4mL) under nitrogen, add Pd(PPh3)4 (0.026 mmol). Stir the mixture at 800C for 16 hours, concentrate, and extract with EtOAc. Dry over Na2SO4, concentrate under vacuum, and purify by preparative TLC (9:1 hexanes/EtOAc) to give 2,3-dichloro-4-(3-chloro-4-fluoro-phenyl)-pyridine.
2. 4-[6-chloro-4-(3-chloro-4-fluoro-phenyl)-pyridin-2-yl]-morpholine
Figure imgf000059_0001
Stir a solution of 2,6-dichloro-4-(3-chloro-4-fluoro-phenyl)-pyridine (100 mg) in morpholine (2 mL) 3 hours at 800C, concentrate, partition between H2O and EtOAc, dry over Na2SO4, and concentrate under vacuum. Purify by preparative TLC (3:1 hexanes/EtOAc) to give 4-[6-chloro-4-(3-chloro-4-fluoro-phenyl)-pyridin-2-yl]-morpholine.
3. 4-{4-(3-Chloro-4-fluoro-phenyl)-6- [4-(l -methyl- lH-imidazol-2-yl)-piperazin-l-yl]~pyridin-2- ylj-morpholine
Figure imgf000059_0002
To a de-gassed mixture of 4-[6-chloro-4-(3-chloro-4-fluoro-phenyl)-pyridin-2-yl]- morpholine (50 mg, 0.153 mmol)), l-(lH-imidazol-2-yl)-piperazine (0.183 mmol), and IM (THF) /-BuOK (0.183 mmol), in toluene (3mL) under nitrogen, add Pd2(dba)3 (0.006 mmol) and BINAP (0.008 mmol). Stir the mixture at 8O0C overnight, concentrate, extract with EtOAc. Dry over Na2SO4, concentrate under vacuum, and purify by preparative TLC (MeOH/DCM eluent) to give 4-{4-(3-chloro-4-fluoro-phenyl)-6-[4-(l-methyl-lH-imidazol-2-yl)-piperazin-l-yl]-pyridin-2-yl}- morpholine. EXAMPLE 3 Additional Representative Heteroaryl Substituted Piperazinyl-Pyridine Analogues
Using routine modifications, the starting materials may be varied and additional steps employed to produce other compounds provided herein. Compounds listed in Table I are prepared using such methods.
Table I
Figure imgf000060_0001
Figure imgf000061_0001
1 -
Figure imgf000062_0001
Figure imgf000063_0001
Figure imgf000064_0001
Figure imgf000065_0001
Figure imgf000066_0001
EXAMPLE 4
VRl-Transfected Cells and Membrane Preparations This Example illustrates the preparation of VRl-transfected cells and VRl -containing membrane preparations for use in capsaicin binding assays (Example 5).
A cDNA encoding full length human capsaicin receptor (SEQ ID NO:1, 2 or 3 of U.S. Patent No. 6,482,611) is subcloned in the plasmid pBK-CMV (Stratagene, La MIa, CA) for recombinant expression in mammalian cells. Human embryonic kidney (HEK293) cells are transfected with the pBK-CMV expression construct encoding the full length human capsaicin receptor using standard methods. The transfected cells are selected over two weeks in media containing G418 (400 μg/ml) to obtain a pool of stably transfected cells. Independent clones are isolated from this pool by limiting dilution to obtain clonal stable cell lines for use in subsequent experiments. For radioligand binding experiments, cells are seeded in T 175 cell culture flasks in media without antibiotics and grown to approximately 90% confluency. The flasks are then washed with PBS and harvested in PBS containing 5 mM EDTA. The cells are pelleted by gentle centrifugation and stored at -800C until assayed.
Previously frozen cells are disrupted with the aid of a tissue homogenizer in ice-cold HEPES homogenization buffer (5mM KCl 5, 5.8mM NaCl, 0.75mM CaCl2, 2mM MgCl2, 320 mM sucrose, and 10 mM HEPES pH 7.4). Tissue homogenates are first centrifuged for 10 minutes at 1000 x g (4°C) to remove the nuclear fraction and debris, and then the supernatant from the first centrifugation is further centrifuged for 30 minutes at 35,000 x g (4°C) to obtain a partially purified membrane fraction. Membranes are resuspended in the HEPES homogenization buffer prior to the assay. An aliquot of this membrane homogenate is used to determine protein concentration via the Bradford method (BIO-RAD Protein Assay Kit, #500-0001, BIO-RAD, Hercules, CA). EXAMPLE 5 Capsaicin Receptor Binding Assay
This Example illustrates a representative assay of capsaicin receptor binding that may be used to determine the binding affinity of compounds for the capsaicin (VRl) receptor. Binding studies with [3H] Resiniferatoxin (RTX) are carried out essentially as described by Szallasi and Blumberg (1992) J. Pharmacol. Exp. Ter. 2(52: 883-888. In this protocol, nonspecific RTX binding is reduced by adding bovine alpha] acid glycoprotein (100 μg per tube) after the binding reaction has been terminated.
[3H] RTX (37 Ci/mmol) is synthesized by and obtained from the Chemical Synthesis and Analysis Laboratory, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD. [3H] RTX may also be obtained from commercial vendors (e.g., Amersham Pharmacia Biotech, Inc.; Piscataway, NJ).
The membrane homogenate of Example 4 is centrifuged as before and resuspended to a protein concentration of 333μg/ml in homogenization buffer. Binding assay mixtures are set up on ice and contain [3H]RTX (specific activity 2200 mCi/ml), 2 μl non-radioactive test compound, 0.25 mg/ml bovine serum albumin (Cohn fraction V), and 5 x 104 - 1 x 105 VRl-transfected cells. The final volume is adjusted to 500 μl (for competition binding assays) or 1,000 μl (for saturation binding assays) with the ice-cold HEPES homogenization buffer solution (pH 7.4) described above. Non-specific binding is defined as that occurring in the presence of 1 μM non-radioactive RTX (Alexis Corp.; San Diego, CA). For saturation binding, [3H]RTX is added in the concentration range of 7-1,000 pM, using 1 to 2 dilutions. Typically 11 concentration points are collected per saturation binding curve.
Competition binding assays are performed in the presence of 60 pM [3H]RTX and various concentrations of test compound. The binding reactions are initiated by transferring the assay mixtures into a 37°C water bath and are terminated following a 60 minute incubation period by cooling the tubes on ice. Membrane-bound RTX is separated from free, as well as any alpha! -acid glycoprotein-bound RTX, by filtration onto WALLAC glass fiber filters (PERKIN-ELMER, Gaithersburg, MD) which were pre-soaked with 1.0% PEI (polyethyleneimine) for 2 hours prior to use. Filters are allowed to dry overnight then counted in a WALLAC 1205 BETA PLATE counter after addition of WALLAC BETA SCINT scintillation fluid.
Equilibrium binding parameters are determined by fitting the allosteric Hill equation to the measured values with the aid of the computer program FIT P (Biosoft, Ferguson, MO) as described by Szallasi, et al. (1993) J. Pharmacol. Exp. Ther. 266:678-683. Compounds provided herein generally exhibit K, values for capsaicin receptor of less than 1 μM, 100 nM, 50 nM, 25 nM, 10 nM, or InM in this assay. EXAMPLE 6 Calcium Mobilization Assay
This Example illustrates representative calcium mobilization assays for use in evaluating test compounds for agonist and antagonist activity. Cells transfected with expression plasmids (as described in Example 4) and thereby expressing human capsaicin receptor are seeded and grown to 70-90% confluency in FALCON black-walled, clear-bottomed 96-well plates (#3904, BECTON-DICKINSON, Franklin Lakes, NJ). The culture medium is emptied from the 96 well plates and FLUO-3 AM calcium sensitive dye (Molecular Probes, Eugene, OR) is added to each well (dye solution: 1 mg FLUO-3 AM, 440 μL DMSO and 440 μl 20% pluronic acid in DMSO, diluted 1 :250 in Krebs-Ringer HEPES (KRH) buffer (25 mM HEPES, 5 mM KCl, 0.96 mM NaH2PO4, 1 mM MgSO4, 2 mM CaCl2, 5 mM glucose, 1 mM probenecid, pH 7.4), 50 μl diluted solution per well). Plates are covered with aluminum foil and incubated at 370C for 1-2 hours in an environment containing 5% CO2. After the incubation, the dye is emptied from the plates, and the cells are washed once with KRH buffer, and resuspended in KRH buffer.
DETERMINATION CAPSAICIN EC50
To measure the ability .of a test compound to agonize or antagonize a calcium mobilization response in cells expressing capsaicin receptors to capsaicin or other vanilloid agonist, the EC50 of the agoϊiist capsaicin is first determined. An additional 20 μl of KRH buffer and 1 μl DMSO is added to each well of cells, prepared as described above. 100 μl capsaicin in KRH buffer is automatically transferred by the FLIPR instrument to each well. Capsaicin-induced calcium mobilization is monitored using either FLUOROSKAN ASCENT (Labsystems; Franklin, MA) or FLIPR (fluorometric imaging plate reader system; Molecular Devices, Sunnyvale, CA) instruments. Data obtained between 30 and 60 seconds after agonist application are used to generate an 8-point concentration response curve, with final capsaicin concentrations of 1 nM to 3 μM. KALEIDAGRAPH software (Synergy Software, Reading, PA) is used to fit the data to the equation: y=a*(l/(l+(b/x)c)) to determine the 50% excitatory concentration (EC50) for the response. In this equation, y is the maximum fluorescence signal, x is the concentration of the agonist or antagonist (in this case, capsaicin), a is the Em3x, b corresponds to the EC50 value and c is the Hill coefficient.
DETERMINATION OF AGONIST ACTIVITY
Test compounds are dissolved in DMSO, diluted in KRH buffer, and immediately added to cells prepared as described above. 100 nM capsaicin (an approximate EC90 concentration) is also added to cells in the same 96-well plate as a positive control. The final concentration of test compounds in the assay wells is between 0.1 nM and 5 μM.
The ability of a test compound to act as an agonist of the capsaicin receptor is determined by measuring the fluorescence response of cells expressing capsaicin receptors elicited by the compound as function of compound concentration. This data is fit as described above to obtain the EC50, which is generally less than 1 micromolar, preferably less than 100 nM, and more preferably less than 10 nM. The extent of efficacy of each test compound is also determined by calculating the response elicited by a concentration of test compound (typically 1 μM) relative to the response elicited by 100 nM capsaicin. This value, called Percent of Signal (POS), is calculated by the following equation:
POS=100*test compound response /100 nM capsaicin response
This analysis provides quantitative assessment of both the potency and efficacy of test compounds as human capsaicin receptor agonists. Agonists of the human capsaicin receptor generally elicit detectable responses at concentrations less than 100 μM, or preferably at concentrations less than 1 μM, or most preferably at concentrations less than 10 nM. Extent of efficacy at human capsaicin receptor is preferably greater than 30 POS, more preferably greater than 80 POS at a concentration of 1 μM. Certain agonists are essentially free of antagonist activity as demonstrated by the absence of detectable antagonist activity in the assay described below at compound concentrations below 4 nM, more preferably at concentrations below 10 μM and most preferably at concentrations less than or equal to 100 μM.
DETERMINATION OF ANTAGONIST ACTIVITY
Test compounds are dissolved in DMSO, diluted in 20 μl KRH buffer so that the final concentration of test compounds in the assay well is between 1 μM and 5 μM, and added to cells prepared as described above. The 96 well plates containing prepared cells and test compounds are incubated in the dark, at room temperature for 0.5 to 6 hours. It is important that the incubation not continue beyond 6 hours. Just prior to determining the fluorescence response, 100 μl capsaicin in KRH buffer at twice the EC50 concentration determined as described above is automatically added by the FLIPR instrument to each well of the 96 well plate for a final sample volume of 200 μl and a final capsaicin concentration equal to the EC50. The final concentration of test compounds in the assay wells is between 1 μM and 5 μM. Antagonists of the capsaicin receptor decrease this response by at least about 20%, preferably by at least about 50%, and most preferably by at least 80%, as compared to matched control (i.e., cells treated with capsaicin at twice the EC50 concentration in the absence of test compound), at a concentration of 10 micromolar or less, preferably 1 micromolar or less. The concentration of antagonist required to provide a 50% decrease, relative to the response observed in the presence of capsaicin and without antagonist, is the IC50 for the antagonist, and is preferably below 1 micromolar, 100 nanomolar, 10 nanomolar or 1 nanomolar.
Certain preferred VRl modulators are antagonists that are essentially free of agonist activity as demonstrated by the absence of detectable agonist activity in the assay described above at compound concentrations below 4 nM, more preferably at concentrations below 10 μM and most preferably at concentrations less than or equal to 100 μM.
EXAMPLE 7 Microsomal in vitro half-life
This Example illustrates the evaluation of compound half-life values (t1/2 values) using a representative liver microsomal half-life assay.
Pooled human liver microsomes are obtained from XenoTech LLC (Kansas City, KS). Such liver microsomes may also be obtained from In Vitro Technologies (Baltimore, MD) or Tissue Transformation Technologies (Edison, NJ). Six test reactions are prepared, each containing 25 μl microsomes, 5 μl of a 100 μM solution of test compound, and 399 μl 0.1 M phosphate buffer (19 niL 0.1 M NaH2PO4, 81 mL 0.1 M Na2HPO4, adjusted to pH 7.4 with H3PO4). A seventh reaction is prepared as a positive control containing 25 μl microsomes, 399 μl 0.1 M phosphate buffer, and 5 μl of a 100 μM solution of a compound with known metabolic properties (e.g., DIAZEPAM or CLOZAPINE). Reactions are preincubated at 390C for 10 minutes.
CoFactor Mixture is prepared by diluting 16.2 mg NADP and 45.4 mg Glucose-6- phosphate in 4 mL 100 mM MgCl2. Glucose-6-phosphate dehydrogenase solution is prepared by diluting 214.3 μl glucose-6-phosphate dehydrogenase suspension (Roche Molecular Biochemicals; Indianapolis, IN) into 1285.7 μl distilled water. 71 μl Starting Reaction Mixture (3 mL CoFactor Mixture; 1.2 mL Glucose-6-phosphate dehydrogenase solution) is added to 5 of the 6 test reactions and to the positive control. 71 μl 100 mM MgCl2 is added to the sixth test reaction, which is used as a negative control. At each time point (0, 1, 3, 5, and 10 minutes), 75 μl of each reaction mix is pipetted into a well of a 96-well deep-well plate containing 75 μl ice-cold acetonitrile. Samples are vortexed and centrifuged 10 minutes at 3500 rpm (Sorval T 6000D centrifuge, HlOOOB rotor). 75 μl of supernatant from each reaction is transferred to a well of a 96-well plate containing 150 μl of a 0.5 μM solution of a compound with a known LCMS profile (internal standard) per well. LCMS analysis of each sample is carried out and the amount of unmetabolized test compound is measured as AUC, compound concentration vs. time is plotted, and the ty2 value of the test compound is extrapolated.
Preferred compounds provided herein exhibit in vitro t]/2 values of greater than 10 minutes and less than 4 hours, preferably between 30 minutes and 1 hour, in human liver microsomes. EXAMPLE 8 MDCK Toxicity Assay
This Example illustrates the evaluation of compound toxicity using a Madin Darby canine kidney (MDCK) cell cytotoxicity assay. 1 μL of test compound is added to each well of a clear bottom 96-well plate (PACKARD,
Meriden, CT) to give final concentration of compound in the assay of 10 micromolar, 100 micromolar or 200 micromolar. Solvent without test compound is added to control wells.
MDCK cells, ATCC no. CCL-34 (American Type Culture Collection, Manassas, VA), are maintained in sterile conditions following the instructions in the ATCC production information sheet. Confluent MDCK cells are trypsinized, harvested, and diluted to a concentration of 0.1 x 106 cells/ml with warm (37°C) medium (VITACELL Minimum Essential Medium Eagle, ATCC catalog # 30-2003). 100 μL of diluted cells is added to each well, except for five standard curve control wells that contain 100 μL of warm medium without cells. The plate is then incubated at 37°C under 95% O2, 5% CO2 for 2 hours with constant shaking. After incubation, 50 μL of mammalian cell lysis solution (from the PACKARD (Meriden, CT) ATP-LITE-M Luminescent ATP detection kit) is added per well, the wells are covered with PACKARD TOPSEAL stickers, and plates are shaken at approximately 700 rpm on a suitable shaker for 2 minutes.
Compounds causing toxicity will decrease ATP production, relative to untreated cells. The ATP-LITE-M Luminescent ATP detection kit is generally used according to the manufacturer's instructions to measure ATP production in treated and untreated MDCK cells. PACKARD ATP LITE-M reagents are allowed to equilibrate to room temperature. Once equilibrated, the lyophilized substrate solution is reconstituted in 5.5 mL of substrate buffer solution (from kit). Lyophilized ATP standard solution is reconstituted in deionized water to give a 10 mM stock. For the five control wells, 10 μL of serially diluted PACKARD standard is added to each of the standard curve control wells to yield a final concentration in each subsequent well of 200 nM, 100 nM, 50 nM, 25 nM and 12.5 nM. PACKARD substrate solution (50 μL) is added to all wells, which are then covered, and the plates are shaken at approximately 700 rpm on a suitable shaker for 2 minutes. A white PACKARD sticker is attached to the bottom of each plate and samples are dark adapted by wrapping plates in foil and placing in the dark for 10 minutes. Luminescence is then measured at 22°C using a luminescence counter (e.g., PACKARD TOPCOUNT Microplate Scintillation and Luminescence Counter or TECAN SPECTRAFLUOR PLUS), and ATP levels calculated from the standard curve. ATP levels in cells treated with test compound(s) are compared to the levels determined for untreated cells. Cells treated with 10 μM of a preferred test compound exhibit ATP levels that are at least 80%, preferably at least 90%, of the untreated cells. When a 100 μM concentration of the test compound is used, cells treated with preferred test compounds exhibit ATP levels that are at least 50%, preferably at least 80%, of the ATP levels detected in untreated cells.
EXAMPLE 9 Dorsal Root Ganglion Cell Assay This Example illustrates a representative dorsal root ganglian cell assay for evaluating
VRl antagonist or agonist activity of a compound.
DRG are dissected from neonatal rats, dissociated and cultured using standard methods
(Aguayo and White (1992) Brain Research 570:61-67). After 48 hour incubation, cells are washed once and incubated for 30-60 minutes with the calcium sensitive dye Fluo 4 AM (2.5-10 ug/ml; TefLabs, Austin, TX). Cells are then washed once. Addition of capsaicin to the cells results in a
VRl -dependent increase in intracellular calcium levels which is monitored by a change in Fluo-4 fluorescence with a fluorometer. Data are collected for 60-180 seconds to determine the maximum fluorescent signal.
For antagonist assays, various concentrations of compound are added to the cells. Fluorescent signal is then plotted as a function of compound concentration to identify the concentration required to achieve a 50% inhibition of the capsaicin-activated response, or IC50.
Antagonists of the capsaicin receptor preferably have an IC50 below 1 micromolar, 100 nanomolar,
10 nanomolar or 1 nanomolar.
For agonist assays, various concentrations of compound are added to the cells without the addition of capsaicin. Compounds that are capsaicin receptor agonists result in a VRl -dependent increase in intracellular calcium levels which is monitored by a change in Fluo-4 fluorescence with a fluorometer. The EC50, or concentration required to achieve 50% of the maximum signal for a capsaicin-activated response, is preferably below 1 micromolar, below 100 nanomolar or below 10 nanomolar.
EXAMPLE 10 Animal Models for Determining Pain Relief
This Example illustrates representative methods for assessing the degree of pain relief provided by a compound. A. Pain Relief Testing
The following methods may be used to assess pain relief.
MECHANICAL ALLODYNIA
Mechanical allodynia (an abnormal response to an innocuous stimulus) is assessed essentially as described by Chaplan et al. (1994) J. Neurosci. Methods 53:55-63 and TaI and Eliav (1998) Pain 64(3):511-518. A series of von Frey filaments of varying rigidity (typically 8-14 filaments in a series) are applied to the plantar surface of the hind paw with just enough force to bend the filament. The filaments are held in this position for no more than three seconds or until a positive allodynic response is displayed by the rat. A positive allodynic response consists of lifting the affected paw followed immediately by licking or shaking of the paw. The order and frequency with which the individual filaments are applied are determined by using Dixon up-down method. Testing is initiated with the middle hair of the series with subsequent filaments being applied in consecutive fashion, ascending or descending, depending on whether a negative or positive response, respectively, is obtained with the initial filament. Compounds are effective in reversing or preventing mechanical allodynia-like symptoms if rats treated with such compounds require stimulation with a Von Frey filament of higher rigidity strength to provoke a positive allodynic response as compared to control untreated or vehicle treated rats. Alternatively, or in addition, testing of an animal in chronic pain may be done before and after compound administration. In such an assay, an effective compound results in an increase in the rigidity of the filament needed to induce a response after treatment, as compared to the filament that induces a response before treatment or in an animal that is also in chronic pain but is left untreated or is treated with vehicle. Test compounds are administered before or after onset of pain. When a test compound is administered after pain onset, testing is performed 10 minutes to three hours after administration. MECHANICAL HYPERALGESIA
Mechanical hyperalgesia (an exaggerated response to painful stimulus) is tested essentially as described by Koch et al. (1996) Analgesia 2(3): 157-164. Rats are placed in individual compartments of a cage with a warmed, perforated metal floor. Hind paw withdrawal duration (i.e., the amount of time for which the animal holds its paw up before placing it back on the floor) is measured after a mild pinprick to the plantar surface of either hind paw.
Compounds produce a reduction in mechanical hyperalgesia if there is a statistically significant decrease in the duration of hindpaw withdrawal. Test compound may be administered before or after onset of pain. For compounds administered after pain onset, testing is performed 10 minutes to three hours after administration. THERMAL HYPERALGESIA
Thermal hyperalgesia (an exaggerated response to noxious thermal stimulus) is measured essentially as described by Hargreaves et al. (1988) Pain. 32(l):77-88. Briefly, a constant radiant heat source is applied the animals' plantar surface of either hind paw. The time to withdrawal (i.e., the amount of time that heat is applied before the animal moves its paw), otherwise described as thermal threshold or latency, determines the animal's hind paw sensitivity to heat. Compounds produce a reduction in thermal hyperalgesia if there is a statistically significant increase in the time to hindpaw withdrawal (i.e., the thermal threshold to response or latency is increased). Test compound may be administered before or after onset of pain. For compounds administered after pain onset, testing is performed 10 minutes to three hours after administration.
B. Pain Models
Pain may be induced using any of the following methods, to allow testing of analgesic efficacy of a compound. In general, compounds provided herein result in a statistically significant reduction in pain as determined by at least one of the previously described testing methods, using male SD rats and at least one of the following models.
ACUTE INFLAMMATORY PAIN MODEL
Acute inflammatory pain is induced using the carrageenan model essentially as described by Field et al. (1997) Br. J. Pharmacol. 121(8):1513-1522. 100-200 μl of 1-2% carrageenan solution is injected into the rats' hind paw. Three to four hours following injection, the animals' sensitivity to thermal and mechanical stimuli is tested using the methods described above. A test compound (0.01 to 50 mg/kg) is administered to the animal, prior to testing, or prior to injection of carrageenan. The compound can be administered orally or through any parenteral route, or topically on the paw. Compounds that relieve pain in this model result in a statistically significant reduction in mechanical allodynia and/or thermal hyperalgesia. CHRONIC INFLAMMATORY PAIN MODEL
Chronic inflammatory pain is induced using one of the following protocols:
1. Essentially as described by Bertorelli et al. (1999) Br. J. Pharmacol. 128(6): 1252-1258, and Stein et al. (1998) Pharmacol. Biochem. Behav. 31(2):455-51, 200 μl Complete Freund's Adjuvant (0.1 mg heat killed and dried M. Tubei'culosis) is injected to the rats' hind paw: 100 μl into the dorsal surface and 100 μl into the plantar surface.
2. Essentially as described by Abbadie et al (1994) J Neurosci. 14(10):5865-5871 rats are injected with 150 μl of CFA (1.5 mg) in the tibio-tarsal joint.
Prior to injection with CFA in either protocol, an individual baseline sensitivity to mechanical and thermal stimulation of the animals' hind paws is obtained for each experimental animal.
Following injection of CFA, rats are tested for thermal hyperalgesia, mechanical allodynia and mechanical hyperalgesia as described above. To verify the development of symptoms, rats are tested on days 5, 6, and 7 following CFA injection. On day 7, animals are treated with a test compound, morphine or vehicle. An oral dose of morphine of 1-5 mg/kg is suitable as positive control. Typically, a dose of 0.01-50 mg/kg of test compound is used. Compounds can be administered as a single bolus prior to testing or once or twice or three times daily, for several days prior to testing. Drugs are administered orally or through any parenteral route, or applied topically to the animal.
Results are expressed as Percent Maximum Potential Efficacy (MPE). 0% MPE is defined as analgesic effect of vehicle, 100% MPE is defined as an animal's return to pre-CFA baseline sensitivity. Compounds that relieve pain in this model result in a MPE of at least 30%.
CHRONIC NEUROPATHIC PAIN MODEL
Chronic neuropathic pain is induced using the chronic constriction injury (CCI) to the rat's sciatic nerve essentially as described by Bennett and Xie (1988) Pain 33:87-107. Rats are anesthetized (e.g. with an intraperitoneal dose of 50-65 mg/kg pentobarbital with additional doses administered as needed). The lateral aspect of each hind limb is shaved and disinfected. Using aseptic technique, an incision is made on the lateral aspect of the hind limb at the mid thigh level.
The biceps femoris is bluntly dissected and the sciatic nerve is exposed. On one hind limb of each animal, four loosely tied ligatures are made around the sciatic nerve approximately 1-2 mm apart. On the other side the sciatic nerve is not ligated and is not manipulated. The muscle is closed with continuous pattern and the skin is closed with wound clips or sutures. Rats are assessed for mechanical allodynia, mechanical hyperalgesia and thermal hyperalgesia as described above.
Compounds that relieve pain in this model result in a statistically significant reduction in mechanical allodynia, mechanical hyperalgesia and/or thermal hyperalgesia when administered (0.01-50 mg/kg, orally, parenterally or topically) immediately prior to testing as a single bolus, or for several days: once or twice or three times daily prior to testing.

Claims

What is claimed is:
1. A compound of the formula:
Figure imgf000076_0001
or a pharmaceutically acceptable salt thereof, wherein: Aτi is a 5-membered aromatic heterocycle that is substituted with from 0 to 4 substituents independently chosen from Ri; Ar2 is phenyl or a 6-membered aromatic heterocycle, each of which is optionally substituted, and is preferably substituted with from 0 to 4 substituents independently chosen from R2; W is CH or N;
X, Y and Z are independently CRx or N, such that at least one of X, Y and Z is N; Rx is independently chosen at each occurrence from hydrogen, Ci-C4alkyl, amino, cyano and mono- and di-(Ci-C4alkyl)amino; Each R] is independently chosen from:
(a) halogen, cyano and nitro;
(b) groups of the formula -Q-M-Ry; and
(c) groups that are taken together with an adjacent Ri to form a fused 5- to 7-membered carbocyclic or heterocyclic ring that is substituted with from 0 to 4 substituents independently chosen from halogen, cyano, nitro and groups of the formula -Q-M-Ry;
Each Q is independently chosen from C0-C4alkylene;
M is independently selected at each occurrence from a single covalent bond, O, C(=O), OC(=O), C(O)O, 0-C(O)O, S(O)1n, N(R2), C(O)N(Ig, C(=NH)N(R2), N(R8)C(O), N(RZ)C(=NH), N(R2)S(O)n,, S(O)1nN(R2) and N[S(O)1nR2]S(O)1n; wherein m is independently selected at each occurrence from O, 1 and 2; and R2 is independently selected at each occurrence from hydrogen, CrC8alkyl and groups that are taken together with Ry to form an optionally substituted 4- to 7- membered heterocycle; and
Each Ry is independently hydrogen, Ci-C8haloalkyl, optionally substituted Ci-C8alkyl, optionally substituted (C3-C8carbocycle)C0-C4alkyl, optionally substituted (4- to 7-membered heterocycle)Co-C4alkyl, or taken together with R2 to form an optionally substituted 4- to 7- membered heterocycle, wherein each alkyl, carbocycle and heterocycle is preferably substituted with from O to 4 substituents independently selected from hydroxy, halogen, amino, cyano, nitro, oxo, -COOH, aminocarbonyl, Ci-C6alkyl, C3-C7cycloalkyl, C2-C6alkyl ether, CrC6alkanoyl, - SO2(Ci -C6alkyl), -SO2NH2, Ci-C8alkoxy, Ci-C8alkylthio, mono- and di-(d- C6alkyl)aminocarbonyl, mono- and di-(Ci-C6alkyl)amino and phenyl; such that Ry is not hydrogen if Q is Coalkyl and M is a single covalent bond; is:
(a) independently chosen from (i) hydroxy, amino, cyano, halogen, -COOH, -SO2NH2, nitro and aminocarbonyl; and (ii) CrC6alkyl, (C3-C8cycloalkyl)C0-C4alkyl, Ci-Qhaloalkyl, Q- C6alkoxy, CrC6alkylthio, C2-C6alkyl ether, C2-C6alkanoyl, Q-Cβalkoxycarbonyl, C2- C6alkanoyloxy, C3-C6alkanone, mono- and di-(Ci-C6alkyl)aminoC0-C6alkyl, mono- and di- (C3-C8cycloalkyl)aminoCo-C4alkyl, . (4- to 7-membered heterocycle)C0-C4alkyl, Q- C6alkylsulfonyl, mono- and di-(Ci-C6alkyl)aminosulfonyl, and mono- and di-(Q- C6alkyl)aminocarbonyl, each of which is optionally substituted, and is preferably substituted with from O to 4 substituents independently chosen from halogen, hydroxy, cyano, amino, - COOH and oxo; or
(b) taken together with an adjacent R2 to form a fused 5- to 13-membered carbocyclic or heterocyclic group that is optionally substituted, and is preferably substituted with from O to 3 substituents independently chosen from halogen, oxo and Ci-C6alkyl;
R3 is selected from:
(i) hydrogen and halogen;
(ii) Ci-C6alkyl, (C3-C8cycloalkyl)C0-C2alkyl, CrC6haloalkyl and phenylC0-C2alkyl; and (iii) groups of the formula: R5
Λ-N- R6 o orr Λ-W^ R7 wherein:
L is Co-C6alkylene or Q-C6alkyl that is taken together with R5, R6 or R7 to form a 4- to 7- membered heterocycle; W is O, CO, S, SO or SO2; R5 and R6 are:
(a) independently chosen from hydrogen, Q-Q2alkyl, C2-Ci2alkenyl, (C3- C8cycloalkyl)Co-C4alkyl, C2-C6alkanoyl, C,-C6alkylsulfonyl, phenylC0-C6alkyl, (4- to 7- membered heterocycle)C0-C6alkyl and groups that are joined to L to form a 4- to 7- membered heterocycle; or
(b) joined to form a 4- to 12-membered heterocycle; and
R7 is hydrogen, Ci-C]2alkyl, C2-Ci2alkenyl, (C3-C3cycloalkyl)Co-C4alkyl, C2-C6alkanoyl, phenylC0-C6alkyl, (4- to 7-membered heterocycle)C0-C6alkyl or a group that is joined to L to form a 4- to 7-membered heterocycle; wherein each of (ii) and (iii) is optionally substituted, and is preferably substituted with from O to
4 substituents independently chosen from: (1) halogen, hydroxy, amino, cyano, -COOH, -SO2NH2, oxo, nitro and aminocarbonyl; and (2) Ci-C6alkyl, (C3-C8cycloalkyl)Co-C2alkyl, C,-C6haloalkyl, CrC6alkoxy, Q- Cδalkoxycarbonyl, CrC6haloalkyl, Ci-C6alkanoyl, C2-C6alkanoylamino, mono- and di- (CrC6alkyl)aminoC0-C4alkyl, Ci-C6alkylsulfonyl, mono- and di-(Q- C6alkyl)aminosulfonyl, mono- and di-(Ci-C6alkyl)aminocarbonylC0-C4alkyl, phenylC0- C4alkyl and (4- to 7-membered heterocycle)C0-C4alkyl, each of which is substituted with from O to 4 secondary substituents independently chosen from halogen, hydroxy, cyano, oxo, imino, CrC4alkyl, Q-C4alkoxy and CrC4haloalkyl; and
R4 represents from O to 2 substituents that are preferably independently chosen from Ci-C3alkyl, Q- C3haloalkyl and oxo.
2. A compound or salt according to claim 1 , wherein the compound has the formula:
Figure imgf000078_0001
wherein:
D, E and G are independently O, S, N, NRla or CRia; and
Each Rla is independently chosen from:
(a) hydrogen, halogen, cyano and nitro;
(b) groups of the formula -Q-M-Ry; and
(c) groups that are taken together with an adjacent R]a to form a fused 5- to 7-membered carbocyclic or heterocyclic ring that is optionally substituted with from 1 to 4 substituents independently chosen from halogen, cyano, nitro and groups of the formula -Q-M-Ry.
3. A compound or salt according to claim 2, wherein the compound has the formula:
Figure imgf000078_0002
4. A compound or salt according to claim 2, wherein the compound has the formula:
Figure imgf000079_0001
5. A compound or salt according to claim 2, wherein the compound has the formula:
R4 Y Z
wherein D is O or S.
6. A compound or salt according to claim 2, wherein the compound has the formula:
Figure imgf000079_0002
wherein D is O or S.
7. A compound or salt according to claim 2, wherein the compound has the formula:
Figure imgf000079_0003
wherein E is N, O or S.
8. A compound or salt according to claim 2, wherein the compound has the formula:
R Y Z
wherein E is N, O or S. εa
9. A compound or salt according to any one of claims 4-8, wherein Ria is hydrogen, halogen, amino, cyano, Q-C6alkyl, CrC6haloalkyl, CrC6alkoxy, d-Qalkylsulfonyl, or mono- or di- (CrC6alkyl)aminosulfonyl.
10. A compound or salt according to claim 9, wherein RJa is hydrogen, halogen, nitro, cyano, methyl or trifluoromethyl.
11. A compound or salt according to any one of claims 1-10, wherein R3 is a group of the formula:
Figure imgf000080_0001
wherein: L is Co-Cβalkylene or Ci-C6alkyl that is taken together with R5 or R6 to form a 4- to 7-membered heterocycle; and R5 and R6 are:
(a) independently chosen from hydrogen, C)-C6alkyl, C2-C6alkenyl, (C3-C8cycloalkyl)Co-C4alkyl, C2-C4alkanoyl and groups that are joined to L to form a 4- to 7-membered heterocycle; or
(b) joined to form a 4- to 12-membered heterocycloalkyl; each of which alkyl, alkenyl, (cycloalkyl)alkyl, alkanoyl and heterocycloalkyl is substituted with from 0 to 4 substituents independently chosen from (i) halogen, hydroxy, amino, aminocarbonyl, oxo, -COOH and -SO2NH2; and (ii) CrC4alkyl, C5-C7cycloalkyl, C,-C4alkoxy, C2-C4alkanoyl, CrC4haloalkyl, mono- and di-(Ci-C4alkyl)aminoC0-C2alkyl, mono- and di-(Cr C4alkyl)aminocarbonylC0-C2alkyl, phenylC0-C4alkyl and (4- to 7-membered heterocycle)C0- C2alkyl, each of which is substituted with from 0 to 4 secondary substituents independently chosen from halogen, hydroxy, cyano, Q-Gjalkyl, Ci-C4alkoxy and Ci-C4haloalkyl.
12. A compound or salt according to claim 11, wherein R3 is di(CrC4alkyl)amino substituted with from 0 to 4 substituents independently chosen from halogen, hydroxy, amino, oxo, aminocarbonyl, -COOH, -SO2NH2, C,-C4alkyl, C2-C4alkenyl, C5-C7cycloalkyl, CrC4haloalkyl, Q- C4alkoxy, C2-C4alkyl ether, C2-C4alkanoyl, Ci-C4alkylsulfonyl, C2-C4alkanoylamino and mono- and di-(Ci -C4alkyl)amino.
13. A compound or salt according to claim 12, wherein R3 is azetidinyl, pyrrolidinyl, morpholinyl, thiomorpholinyl, piperidinyl, piperazinyl, tetrahydropyridyl or azepanyl, each of which is substituted with from O to 4 substituents independently chosen from:
(a) halogen, hydroxy, amino, oxo, aminocarbonyl, SO2NH2 and -COOH; and (b) Ci-C4alkyl, C2-C4alkenyl, C5-C7cycloalkyl, CrC4haloalkyl, CrC4alkoxy, C2-C4alkyl ether, C2-C4alkanoyl, Ci-C4alkylsulfonyl, C2-C4alkanoylamino and mono- and di-(Cr C4alkyl)amino, each of which is substituted with from O to 4 secondary substituents independently chosen from hydroxy and halogen.
14. A compound or salt according to claim 13, wherein R3 is chosen from:
Figure imgf000081_0001
15. A compound or salt according to any one of claims 1-10, wherein R3 is a group of the formula:
^- K R7 wherein:
L is Co-C6alkylene or Q-Cβalkyl that is taken together with R7 to form a 4- to 7-membered heterocycle;
W is O; and
R7 is hydrogen, Ci-C]2alkyl, C2-Ci2alkenyl, (C3-C8cycloalkyl)C0-C4alkyl, C2-C6alkanoyl, phenylCo-Cfjalkyl, (4- to 7-membered heterocycle)C0-C6alkyl or a group that is joined to L to form a 4- to 7-membered heterocycle, each of which alkyl, alkenyl, (cycloalkyl)alkyl, alkanoyl and heterocycloalkyl is substituted with from 0 to 4 substituents independently chosen from (i) halogen, hydroxy, amino, aminocarbonyl, oxo, -<:OOH and -SO2NH2; and (ii) d-C4alkyl, C3-C7cycloalkyl, C,-C4alkoxy, C2-C4alkanoyl, Ci-Qhaloalkyl, mono- and di-(Ci-C4alkyl)aminoC0-C2alkyl, mono- and di-(Q- C4alkyl)aminocarbonylC0-C2alkyl, ρhenylCo-C4alkyl and (4- to 7-membered heterocycle)Co-C2alkyl, each of which is substituted with from 0 to 4 secondary substituents independently chosen from halogen, hydroxy, cyano, Ci-C4alkyl, Q- C4alkoxy and CrC4haloalkyl.
16. A compound or salt according to any one of claims 1-15, wherein Ar2 is unsubstituted phenyl or unsubstituted pyridyl.
17. A compound or salt according to any one of claims 1-15, wherein Ar2 has at least one substituent chosen from amino, cyano, halogen, -SO2NH2, Ci-C4alkyl, Ci-C4haloalkyl, Q- C4hydroxyalkyl, Q-Qalkoxy, Ci-C4alkylthio, Q-C4haloalkoxy, mono- and di-(Q-Qalkyl)aminoC0- Qalkyl, mono- and di-(C3-C8cycloalkyl)aminoC0-C4alkyl, C2-C4alkanoyl, Q-Qalkoxycarbonyl, Q- C4alkylsulfonyl, Q-Qhaloalkylsulfonyl and mono- and di-(Q-Qalkyl)aminosulfonyl.
18. A compound or salt according to claim 17, wherein Ar2 is phenyl, pyridyl or pyrimidyl, each of which is substituted with from 1 to 3 substituents independently chosen from amino, cyano, halogen, Q-C4alkyl, Q-Qhaloalkyl, Q-C4alkoxy, Q-C4haloalkoxy, Q-C4alkylthio, and mono- and di-(Q-C4alkyl)aminoC0-C4alkyl.
19. A compound or salt according to claim 1, wherein the compound has the formula:
Figure imgf000082_0001
wherein:
D, E and G are independently O, S, N, NRla or CRia;
Each Rla is independently chosen from:
(a) hydrogen, halogen, cyano and nitro; and
(b) groups of the formula -Q-M-Ry;
Ar2 is phenyl, pyridyl or pyrimidyl, each of which is substituted with from O to 3 substituents independently chosen from amino, cyano, halogen, Q-C4alkyl, Q-C4haloalkyl, Q-Qalkoxy, Q- Qhaloalkoxy, Q-C4alkylthio, and mono- and di-(Q-C4alkyl)aminoC0-Qalkyl; and
R4 represents O substituents or one methyl substituent.
20. A compound or salt according to any one of claims 1-19, wherein the group:
Figure imgf000082_0002
21. A compound or salt according to any one of claims 1-20, wherein the compound is a VRl antagonist and has an IC50 value of 1 micromolar or less in a capsaicin receptor calcium mobilization assay.
22. A compound or salt according to claim 21, wherein the compound exhibits no detectable agonist activity in an in vitro assay of capsaicin receptor agonism at a concentration of compound equal to the ICs0.
23. A pharmaceutical composition, comprising at least one compound or salt according to any one of claims 1-22, in combination with a physiologically acceptable carrier or excipient.
24. A pharmaceutical composition according to claim 23, wherein the composition is formulated as an injectible fluid, an aerosol, a cream, a gel, a pill, a capsule, a syrup or a transdermal patch.
25. A method for reducing calcium conductance of a cellular capsaicin receptor, comprising contacting a cell expressing a capsaicin receptor with a compound or salt according to any one of claims 1-22, and thereby reducing calcium conductance of the capsaicin receptor.
26. A method according to claim 25, wherein the cell is contacted in vivo in an animal.
27. A method according to claim 25, wherein the cell is a neuronal cell.
28. A method according to claim 25, wherein the cell is a urothelial cell.
29. A method according to claim 26, wherein during contact the compound is present within a body fluid of the animal.
30. A method according to claim 26, wherein the animal is a human.
31. A method according to claim 26, wherein the compound or salt is administered orally.
32. A method for inhibiting binding of vanilloid ligand to a capsaicin receptor in vitro, the method comprising contacting capsaicin receptor with a compound or salt according to any one of claims 1-22, in an amount sufficient to detectably inhibit vanilloid ligand binding to capsaicin receptor.
33. A method for inhibiting binding of vanilloid ligand to a capsaicin receptor in a patient, the method comprising contacting cells expressing capsaicin receptor with a compound or salt according to any one of claims 1-22, in an amount sufficient to detectably inhibit vanilloid ligand binding to cells expressing a cloned capsaicin receptor in vitro, and thereby inhibiting binding of vanilloid ligand to the capsaicin receptor in the patient.
34. A method according to claim 33, wherein the patient is a human.
35. A method according to claim 33, wherein the compound is present in the blood of the patient at a concentration of 1 micromolar or less.
36. A method for treating a condition responsive to capsaicin receptor modulation in a patient, comprising administering to the patient a therapeutically effective amount of a compound or salt according to any one of claims 1-22, and thereby alleviating the condition in the patient.
37. A method according to claim 36, wherein the patient is suffering from (i) exposure to capsaicin, (ii) burn or irritation due to exposure to heat, (iii) burns or irritation due to exposure to light, (iv) burn, bronchoconstriction or irritation due to exposure to tear gas, infectious agents, air pollutants or pepper spray, or (v) burn or irritation due to exposure to acid.
38. A method according to claim 36, wherein the condition is asthma or chronic obstructive pulmonary disease.
39. A method for treating pain in a patient, comprising administering to a patient suffering from pain a therapeutically effective amount of a compound or salt according to any one of claims 1- 22, and thereby alleviating pain in the patient.
40. A method according to claim 39, wherein the compound or salt is present in the blood of the patient at a concentration of 1 micromolar or less.
41. A method according to claim 39, wherein the patient is suffering from neuropathic pain.
42. A method according to claim 39, wherein the pain is associated with a condition selected from: postmastectomy pain syndrome, stump pain, phantom limb pain, oral neuropathic pain, toothache, postherpetic neuralgia, diabetic neuropathy, reflex sympathetic dystrophy, trigeminal neuralgia, osteoarthritis, rheumatoid arthritis, fibromyalgia, Guillain-Barre syndrome, meralgia paresthetica, burning-mouth syndrome, bilateral peripheral neuropathy, causalgia, neuritis, neuronitis, neuralgia, AIDS-related neuropathy, MS-related neuropathy, spinal cord injury-related pain, surgery- related pain, musculoskeletal pain, back pain, headache, migraine, angina, labor, hemorrhoids, dyspepsia, Charcot's pains, intestinal gas, menstruation, cancer, venom exposure, irritable bowel syndrome, inflammatory bowel disease and trauma.
43. A method according to claim 39, wherein the patient is a human.
44. A method for treating itch in a patient, comprising administering to a patient a therapeutically effective amount of a compound or salt according to any one of claims 1-22, and thereby alleviating itch in the patient.
45. A method for treating cough or hiccup in a patient, comprising administering to a patient a therapeutically effective amount of a compound or salt according to any one of claims 1-22, and thereby alleviating cough or hiccup in the patient.
46. A method for treating urinary incontinence or overactive bladder in a patient, comprising administering to a patient a therapeutically effective amount of a compound or salt according to any one of claims 1-22, and thereby alleviating urinary incontinence or overactive bladder in the patient.
47. A method promoting weight loss in an obese patient, comprising administering to a patient a therapeutically effective amount of a compound or salt according to any one of claims 1-22, and thereby promoting weight loss in the patient.
48. A compound or salt according to any one of claims 1-22, wherein the compound is radiolabeled.
49. A method for identifying an agent that binds to capsaicin receptor, comprising:
(a) contacting capsaicin receptor with a radiolabeled compound or salt according to claim 48, under conditions that permit binding of the VRl modulator to capsaicin receptor, thereby generating bound, labeled VRl modulator;
(b) detecting a- signal that corresponds to the amount of bound, labeled VRl modulator in the absence of test agent;
(c) contacting the bound, labeled VRl modulator with a test agent;
(d) detecting a signal that corresponds to the amount of bound labeled VRl modulator in the presence of test agent; and
(e) detecting a decrease in signal detected in step (d), as compared to the signal detected in step (b), and therefrom identifying an agent that binds to capsaicin receptor.
50. A method for determining the presence or absence of capsaicin receptor in a sample, comprising the steps of:
(a) contacting a sample with a compound according to any one of claims 1-22, under conditions that permit binding of the compound to capsaicin receptor; and
(b) detecting a level of the compound bound to capsaicin receptor, and therefrom determining the presence or absence of capsaicin receptor in the sample.
51. A method according to claim 50, wherein the compound is radiolabeled, and wherein the step of detection comprises the steps of: (i) separating unbound compound from bound compound; and
(ii) detecting the presence or absence of bound compound in the sample.
52. A packaged pharmaceutical preparation, comprising:
(a) a pharmaceutical composition according to claim 23 in a container; and
(b) instructions for using the composition to treat pain.
53. A packaged pharmaceutical preparation, comprising:
(a) a pharmaceutical composition according to claim 23 in a container; and
(b) instructions for using the composition to treat cough or hiccup.
54. A packaged pharmaceutical preparation, comprising:
(a) a pharmaceutical composition according to claim 23 in a container; and
(b) instructions for using the composition to treat obesity.
55. A packaged pharmaceutical preparation, comprising:
(a) a pharmaceutical composition according to claim 23 in a container; and
(b) instructions for using the composition to treat urinary incontinence or overactive bladder.
56. The use of a compound or salt according to any one of claims 1-22 for the manufacture of a medicament for the treatment of a condition responsive to capsaicin receptor modulation.
57. A use according to 56, wherein the condition is pain, asthma, chronic obstructive pulmonary disease, cough, hiccup, obesity, urinary incontinence, overactive bladder, exposure to capsaicin, burn or irritation due to exposure to heat, burn or irritation due to exposure to light, burn, bronchoconstriction or irritation due to exposure to tear gas, infectious agents, air pollutants or pepper spray, or burn or irritation due to exposure to acid.
PCT/US2006/002194 2005-01-19 2006-01-19 Heteroaryl substituted piperazinyl-pyridine analogues WO2006078992A2 (en)

Priority Applications (5)

Application Number Priority Date Filing Date Title
CA002595205A CA2595205A1 (en) 2005-01-19 2006-01-19 Heteroaryl substituted piperazinyl-pyridine analogues
EP06733793A EP1838703A2 (en) 2005-01-19 2006-01-19 Heteroaryl substituted piperazinyl-pyridine analogues
JP2007552316A JP2008528506A (en) 2005-01-19 2006-01-19 Heteroaryl-substituted piperazinyl-pyridine analogs
AU2006206246A AU2006206246A1 (en) 2005-01-19 2006-01-19 Heteroaryl substituted piperazinyl-pyridine analogues
US11/795,651 US20080124384A1 (en) 2005-01-19 2006-01-19 Heteroaryl Substituted Piperazinyl-Pyridine Analogues

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US64549605P 2005-01-19 2005-01-19
US60/645,496 2005-01-19

Publications (2)

Publication Number Publication Date
WO2006078992A2 true WO2006078992A2 (en) 2006-07-27
WO2006078992A3 WO2006078992A3 (en) 2006-09-28

Family

ID=36692967

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2006/002194 WO2006078992A2 (en) 2005-01-19 2006-01-19 Heteroaryl substituted piperazinyl-pyridine analogues

Country Status (7)

Country Link
US (1) US20080124384A1 (en)
EP (1) EP1838703A2 (en)
JP (1) JP2008528506A (en)
CN (1) CN101107245A (en)
AU (1) AU2006206246A1 (en)
CA (1) CA2595205A1 (en)
WO (1) WO2006078992A2 (en)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013040059A1 (en) * 2011-09-12 2013-03-21 Merck Patent Gmbh Novel imidazole amines as modulators of kinase activity
US8865894B2 (en) 2012-02-24 2014-10-21 Novartis Ag Oxazolidin-2-one compounds and uses thereof
US8957068B2 (en) 2011-09-27 2015-02-17 Novartis Ag 3-pyrimidin-4-yl-oxazolidin-2-ones as inhibitors of mutant IDH
US9296733B2 (en) 2012-11-12 2016-03-29 Novartis Ag Oxazolidin-2-one-pyrimidine derivative and use thereof for the treatment of conditions, diseases and disorders dependent upon PI3 kinases
US9321760B2 (en) 2011-09-12 2016-04-26 Merck Patent Gmbh Aminopyrimidine derivatives for use as modulators of kinase activity
US9434719B2 (en) 2013-03-14 2016-09-06 Novartis Ag 3-pyrimidin-4-yl-oxazolidin-2-ones as inhibitors of mutant IDH
US10894787B2 (en) 2010-09-22 2021-01-19 Arena Pharmaceuticals, Inc. Modulators of the GPR119 receptor and the treatment of disorders related thereto
US11007175B2 (en) 2015-01-06 2021-05-18 Arena Pharmaceuticals, Inc. Methods of treating conditions related to the S1P1 receptor
US11534424B2 (en) 2017-02-16 2022-12-27 Arena Pharmaceuticals, Inc. Compounds and methods for treatment of primary biliary cholangitis
US11884626B2 (en) 2015-06-22 2024-01-30 Arena Pharmaceuticals, Inc. Crystalline L-arginine salt of (R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta [b]indol-3-yl)acetic acid(Compound1) for use in S1P1 receptor-associated disorders

Families Citing this family (33)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RS20050532A (en) 2003-01-14 2007-12-31 Arena Pharmaceuticals Inc., 1,2,3-trisubstituted aryl and heteroaryl derivatives as modulators of metabolism and the prpphylaxis and treatment of disorders related thereto such as diabetes and hyperglycemia
AR045047A1 (en) * 2003-07-11 2005-10-12 Arena Pharm Inc ARILO AND HETEROARILO DERIVATIVES TRISUSTITUIDOS AS MODULATORS OF METABOLISM AND PROFILAXIS AND TREATMENT OF DISORDERS RELATED TO THEMSELVES
MXPA06000554A (en) 2003-07-14 2006-07-03 Arena Pharm Inc Fused-aryl and heteroaryl derivatives as modulators of metabolism and the prophylaxis and treatment of disorders related thereto.
AU2004257289A1 (en) * 2003-07-16 2005-01-27 Neurogen Corporation Biaryl piperazinyl-pyridine analogues
MY145822A (en) 2004-08-13 2012-04-30 Neurogen Corp Substituted biaryl piperazinyl-pyridine analogues
MY148521A (en) 2005-01-10 2013-04-30 Arena Pharm Inc Substituted pyridinyl and pyrimidinyl derivatives as modulators of metabolism and the treatment of disorders related thereto
WO2007041358A2 (en) * 2005-09-30 2007-04-12 Miikana Therapeutics, Inc. Substituted pyrazole compounds
WO2008106128A2 (en) * 2007-02-26 2008-09-04 Vitae Pharmaceuticals, Inc. CYCLIC UREA AND CARBAMATE INHIBITORS OF 11β -HYDROXYSTEROID DEHYDROGENASE 1
AU2008262291A1 (en) * 2007-06-11 2008-12-18 Miikana Therapeutics, Inc. Substituted pyrazole compounds
JP5451611B2 (en) * 2007-07-26 2014-03-26 ヴァイティー ファーマシューティカルズ，インコーポレイテッド Cyclic inhibitor of 11β-hydroxysteroid dehydrogenase 1
AR069207A1 (en) * 2007-11-07 2010-01-06 Vitae Pharmaceuticals Inc CYCLIC UREAS AS INHIBITORS OF THE 11 BETA - HIDROXI-ESTEROIDE DESHIDROGENASA 1
US8440658B2 (en) 2007-12-11 2013-05-14 Vitae Pharmaceuticals, Inc. Cyclic urea inhibitors of 11β-hydroxysteroid dehydrogenase 1
TW200934490A (en) * 2008-01-07 2009-08-16 Vitae Pharmaceuticals Inc Lactam inhibitors of 11 &abgr;-hydroxysteroid dehydrogenase 1
WO2009094169A1 (en) * 2008-01-24 2009-07-30 Vitae Pharmaceuticals, Inc. Cyclic carbazate and semicarbazide inhibitors of 11beta-hydroxysteroid dehydrogenase 1
WO2009102428A2 (en) * 2008-02-11 2009-08-20 Vitae Pharmaceuticals, Inc. 1,3-OXAZEPAN-2-ONE AND 1,3-DIAZEPAN-2-ONE INHIBITORS OF 11β -HYDROXYSTEROID DEHYDROGENASE 1
EP2254872A2 (en) * 2008-02-15 2010-12-01 Vitae Pharmaceuticals, Inc. Cycloalkyl lactame derivatives as inhibitors of 11-beta-hydroxysteroid dehydrogenase 1
WO2009117109A1 (en) * 2008-03-18 2009-09-24 Vitae Pharmaceuticals, Inc. Inhibitors of 11beta-hydroxysteroid dehydrogenase type 1
EP2291373B1 (en) * 2008-05-01 2013-09-11 Vitae Pharmaceuticals, Inc. Cyclic inhibitors of 11beta-hydroxysteroid dehydrogenase 1
US8569292B2 (en) 2008-05-01 2013-10-29 Vitae Pharmaceuticals, Inc. Cyclic inhibitors of 11β-hydroxysteroid dehydrogenase 1
RU2531272C2 (en) * 2008-05-01 2014-10-20 Вайтаи Фармасьютиклз, Инк. Cyclic inhibitors of 11beta-hydroxysteroid-dehydrogenase 1
CA2723034A1 (en) 2008-05-01 2009-11-05 Vitae Pharmaceuticals, Inc. Cyclic inhibitors of 11beta-hydroxysteroid dehydrogenase 1
JP5777030B2 (en) 2008-07-25 2015-09-09 ベーリンガー インゲルハイム インターナショナル ゲゼルシャフト ミット ベシュレンクテル ハフツング Inhibitor of 11β-hydroxysteroid dehydrogenase 1
WO2010127237A2 (en) * 2009-04-30 2010-11-04 Boehringer Ingelheim International Gmbh Cyclic inhibitors of 11beta-hydroxysteroid dehydrogenase 1
EP2324018B1 (en) 2008-07-25 2013-09-04 Boehringer Ingelheim International GmbH Cyclic inhibitors of 11 beta-hydroxysteroid dehydrogenase 1
US8637505B2 (en) 2009-02-04 2014-01-28 Boehringer Ingelheim International Gmbh Cyclic inhibitors of 11beta-hydroxysteroid dehydrogenase 1
TW201039034A (en) * 2009-04-27 2010-11-01 Chunghwa Picture Tubes Ltd Pixel structure and the method of forming the same
UA109255C2 (en) 2009-04-30 2015-08-10 Берінгер Інгельхайм Інтернешнл Гмбх Cyclic inhibitors of 11beta-hydroxysteroid dehydrogenase 1
US8927539B2 (en) 2009-06-11 2015-01-06 Vitae Pharmaceuticals, Inc. Cyclic inhibitors of 11β-hydroxysteroid dehydrogenase 1 based on the 1,3-oxazinan-2-one structure
JP5749263B2 (en) 2009-07-01 2015-07-15 ヴァイティー ファーマシューティカルズ，インコーポレイテッド Cyclic inhibitor of 11β-hydroxysteroid dehydrogenase 1
US8933072B2 (en) 2010-06-16 2015-01-13 Vitae Pharmaceuticals, Inc. Substituted 5-,6- and 7-membered heterocycles, medicaments containing such compounds, and their use
EP2585444B1 (en) 2010-06-25 2014-10-22 Boehringer Ingelheim International GmbH Azaspirohexanones as inhibitors of 11-beta-hsd1 for the treatment of metabolic disorders
EP2635268A1 (en) 2010-11-02 2013-09-11 Boehringer Ingelheim International GmbH Pharmaceutical combinations for the treatment of metabolic disorders
WO2019193516A2 (en) * 2018-04-04 2019-10-10 Janssen Pharmaceutica Nv Substituted pyridine and pyrimidines and their use as glun2b receptor modulators

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060122394A1 (en) * 2004-08-13 2006-06-08 Blum Charles A Substituted biaryl piperazinyl-pyridine analogues

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060122934A1 (en) * 2004-12-07 2006-06-08 Ncr Corporation Radio frequency identification (RFID) system

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060122394A1 (en) * 2004-08-13 2006-06-08 Blum Charles A Substituted biaryl piperazinyl-pyridine analogues

Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10894787B2 (en) 2010-09-22 2021-01-19 Arena Pharmaceuticals, Inc. Modulators of the GPR119 receptor and the treatment of disorders related thereto
US9662330B2 (en) 2011-09-12 2017-05-30 Merck Patent Gmbh Aminopyrimidine derivatives for use as modulators of kinase activity
KR20140063694A (en) * 2011-09-12 2014-05-27 메르크 파텐트 게엠베하 Novel imidazole amines as modulators of kinase activity
CN104080782A (en) * 2011-09-12 2014-10-01 默克专利有限公司 Novel imidazole amines as modulators of kinase activity
US9145392B2 (en) 2011-09-12 2015-09-29 Merck Patent Gmbh Imidazole amines as modulators of kinase activity
KR101995605B1 (en) 2011-09-12 2019-07-02 메르크 파텐트 게엠베하 Novel Imidazole Amines as Modulators of Kinase Activity
US9321760B2 (en) 2011-09-12 2016-04-26 Merck Patent Gmbh Aminopyrimidine derivatives for use as modulators of kinase activity
CN104080782B (en) * 2011-09-12 2016-06-01 默克专利有限公司 As the aminoimidazole of modulators of kinase activity
US10080750B2 (en) 2011-09-12 2018-09-25 Merck Patent Gmbh Aminopyrimidine derivatives for use as modulators of kinase activity
WO2013040059A1 (en) * 2011-09-12 2013-03-21 Merck Patent Gmbh Novel imidazole amines as modulators of kinase activity
EA025098B1 (en) * 2011-09-12 2016-11-30 Мерк Патент Гмбх Imidazole amines as modulators of kinase activity
US8957068B2 (en) 2011-09-27 2015-02-17 Novartis Ag 3-pyrimidin-4-yl-oxazolidin-2-ones as inhibitors of mutant IDH
US9458177B2 (en) 2012-02-24 2016-10-04 Novartis Ag Oxazolidin-2-one compounds and uses thereof
US8865894B2 (en) 2012-02-24 2014-10-21 Novartis Ag Oxazolidin-2-one compounds and uses thereof
US10202371B2 (en) 2012-11-12 2019-02-12 Novartis Ag Oxazolidin-2-one-pyrimidine derivatives and the use thereof as phosphatidylinositol-3-kinase inhibitors
US9296733B2 (en) 2012-11-12 2016-03-29 Novartis Ag Oxazolidin-2-one-pyrimidine derivative and use thereof for the treatment of conditions, diseases and disorders dependent upon PI3 kinases
US9688672B2 (en) 2013-03-14 2017-06-27 Novartis Ag 3-pyrimidin-4-yl-oxazolidin-2-ones as inhibitors of mutant IDH
US9434719B2 (en) 2013-03-14 2016-09-06 Novartis Ag 3-pyrimidin-4-yl-oxazolidin-2-ones as inhibitors of mutant IDH
US10112931B2 (en) 2013-03-14 2018-10-30 Novartis Ag 3-pyrimidin-4-yl-oxazolidin-2-ones as inhibitors of mutant IDH
US11007175B2 (en) 2015-01-06 2021-05-18 Arena Pharmaceuticals, Inc. Methods of treating conditions related to the S1P1 receptor
US11884626B2 (en) 2015-06-22 2024-01-30 Arena Pharmaceuticals, Inc. Crystalline L-arginine salt of (R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta [b]indol-3-yl)acetic acid(Compound1) for use in S1P1 receptor-associated disorders
US11534424B2 (en) 2017-02-16 2022-12-27 Arena Pharmaceuticals, Inc. Compounds and methods for treatment of primary biliary cholangitis

Also Published As

Publication number Publication date
JP2008528506A (en) 2008-07-31
US20080124384A1 (en) 2008-05-29
WO2006078992A3 (en) 2006-09-28
AU2006206246A1 (en) 2006-07-27
CN101107245A (en) 2008-01-16
EP1838703A2 (en) 2007-10-03
CA2595205A1 (en) 2006-07-27

Similar Documents

Publication Publication Date Title
WO2006078992A2 (en) Heteroaryl substituted piperazinyl-pyridine analogues
EP1786800B1 (en) 1,4-Diarylpiperazine derivatives as capsaicin receptor modulators for treating pain
US20060194805A1 (en) Capsaicin receptor agonists
US20070043049A1 (en) Substituted heterocyclic diarylamine analogues
US20060089354A1 (en) Carboxylic acid, phosphate or phosphonate substituted quinazolin-4-ylamine analogues as capsaicin receptor modulators
US20080175794A1 (en) Substituted Pyridazinyl- and Pyrimidinyl-Quinolin-4-Ylamine Analogues
US20080085901A1 (en) Heteroaryl Substituted Quinolin-4-Ylamine Analogues
US20070191374A1 (en) Substituted cinnolin-4-ylamines
WO2006042289A2 (en) Substituted biaryl quinolin-4-ylamine analogues
US20070219203A1 (en) Arylalkylamino-substituted quinazoline analogues
EP1678173A2 (en) 4-heterobicyclylamino-substituted quinazolines and analogues thereof as capsaicin-antagonists
US20080132509A1 (en) Substituted Biaryl Analogues
US20080045525A1 (en) Piperazinyl-Pyridine Analogues
AU2005218615A1 (en) Heteroalkyl-substituted biphenyl-4-carboxylic acid arylamide analogues
US20070203133A1 (en) Substituted 5,12-diaza-benzoanthracene analogues
EP1648892B1 (en) Aryl-substituted benzo[d]isothiazol-3-ylamine analogues as capsaicin receptor modulators
WO2009100403A1 (en) Substituted aryl pyrimidinones
AU2005280366B2 (en) Substituted biaryl piperazinyl-pyridine analogues

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 200680002730.9

Country of ref document: CN

121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 2006206246

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 2006733793

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 2006206246

Country of ref document: AU

Date of ref document: 20060119

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2595205

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 11795651

Country of ref document: US

Ref document number: 2007552316

Country of ref document: JP

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 6413/DELNP/2007

Country of ref document: IN